Combined methods and compositions for coagulation and tumor treatment

ABSTRACT

Disclosed are various compositions and methods for use in achieving specific blood coagulation. This is exemplified by the specific in vivo coagulation of tumor vasculature, causing tumor regression, through the site-specific delivery of a coagulant using a bispecific antibody.

[0001] The present application is a continuation-in-part of co-pendingU.S. patent application Ser. No. 08/273,567, filed Jun. 11, 1994; whichis a continuation-in-part of co-pending U.S. patent application Ser. No.08/205,330,.filed, Mar. 2, 1994; which is a continuation-in-part of U.S.Ser. No. 07/846,349, filed Mar. 5, 1992. The entire text and figures ofthe above-referenced disclosures are specifically incorporated herein byreference without disclaimer.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates generally to the fields of bloodvessels and of coagulation. More particularly, it provides a variety ofgrowth factor-based and immunological reagents, including bispecificantibodies, for use in achieving specific coagulation.

[0004] 2. Description of the Related Art

[0005] Advances in the chemotherapy of neoplastic disease have beenrealized during the last 30 years. This includes some progress in thedevelopment of new chemotherapeutic agents and, more particularly, thedevelopment of regimens for concurrent administration of drugs. Asignificant understanding of the neoplastic processes at the cellularand tissue level, and the mechanism of action of basic antineoplasticagents, has also allowed advances in the chemotherapy of a number ofneoplastic diseases, including choriocarcinoma, Wilm's tumor, acuteleukemia, rhabdomyosarcoma, retinoblastoma, Hodgkin's disease andBurkitt's lymphoma. Despite the advances that have been made in a fewtumors, though, many of the most prevalent forms of human cancer stillresist effective chemotherapeutic intervention.

[0006] A significant underlying problem that must be addressed in anytreatment regimen is the concept of “total cell kill.” This conceptholds that in order to have an effective treatment regimen, whether itbe a surgical or chemotherapeutic approach or both, there must be atotal cell kill of all so-called “clonogenic” malignant cells, that is,cells that have the ability to grow uncontrolled and replace any tumormass that might be removed. Due to the ultimate need to developtherapeutic agents and regimens that will achieve a total cell kill,certain types of tumors have been more amenable than others to therapy.For example, the soft tissue tumors (e.g., lymphomas), and tumors of theblood and blood-forming organs (e.g., leukemias) have generally beenmore responsive to chemotherapeutic therapy than have solid tumors suchas carcinomas.

[0007] One reason for the susceptibility of soft and blood-based tumorsto chemotherapy is the greater physical accessibility of lymphoma andleukemic cells to chemotherapeutic intervention. Simply put, it is muchmore difficult for most chemotherapeutic agents to reach all of thecells of a solid tumor mass than it is the soft tumors and blood-basedtumors, and therefore much more difficult to achieve a total cell kill.Increasing the dose of chemotherapeutic agents most often results intoxic side effects, which generally limits the effectiveness ofconventional anti-tumor agents.

[0008] The strategy to develop successful antitumor agents involves thedesign of agents that will selectively kill tumor cells, while exertingrelatively little, if any, untoward effects against normal tissues. Thisgoal has been elusive to achieve, though, in that there are fewqualitative differences between neoplastic and normal tissues. Becauseof this, much research over the years has focused on identifyingtumor-specific “marker antigens” that can serve as immunological targetsboth for chemotherapy and diagnosis. Many tumor-specific, orquasi-tumor-specific (“tumor-associated”), markers have been identifiedas tumor cell antigens that can be recognized by specific antibodies.Unfortunately, it is generally the case that tumor specific antibodieswill not in and of themselves exert sufficient antitumor effects to makethem useful in cancer therapy.

[0009] More recently, immunotoxins have been employed in an attempt toselectively target cancer cells. Immunotoxins are conjugates of aspecific targeting agent, typically a tumor-directed antibody orfragment, with a cytotoxic agent, such as a toxin moiety. The targetingagent is designed to direct the toxin to cells carrying the targetedantigen and to kill such cells. “Second generation” immunotoxins havenow been developed, for example, those that employ deglycosylated ricinA chain to prevent entrapment of the immunotoxin by the liver and reducehepatotoxicity (Blakey et al., 1987a;b), and those with new crosslinkersto endow the immunotoxins with higher in vivo stability (Thorpe et al.,1988).

[0010] Immunotoxins have proven effective at treating lymphomas andleukemias in mice (Thorpe et al., 1988; Ghetie et al., 1991; Griffin etal., 1988a;b) and in man (Vitetta et al., 1991). However, lymphoidneoplasias are particularly amenable to immunotoxin therapy because thetumor cells are relatively accessible to blood-borne immunotoxins. Also,it is possible to target normal lymphoid antigens because the normallymphocytes, which are killed along with the malignant cells duringtherapy, are rapidly regenerated from progenitors lacking the targetantigens.

[0011] In contrast with their efficacy in lymphomas, immunotoxins haveproved relatively ineffective in the treatment of solid tumors (Weineret al., 1989; Byers et al., 1989). The principal reason for this is thatsolid tumors are generally impermeable to antibody-sized molecules:specific uptake values of less than 0.001% of the injected dose/g oftumor are not uncommon in human studies (Sands et al., 1988; Epenetos etal., 1986). Another significant problem is that antigen-deficientmutants can escape being killed by the immunotoxin and regrow (Thorpe etal., 1988).

[0012] Furthermore, antibodies that enter the tumor mass do notdistribute evenly for several reasons. Firstly, the dense packing oftumor cells and fibrous tumor stromas present a formidable physicalbarrier to macromolecular transport and, combined with the absence oflymphatic drainage, create an elevated interstitial pressure in thetumor core which reduces extravasation and fluid convection (Baxter etal., 1991; Jain, 1990). Secondly, the distribution of blood vessels inmost tumors is disorganized and heterogeneous, so some tumor cells areseparated from extravasating antibody by large diffusion distances(Jain, 1990). Thirdly, all of the antibody entering the tumor may becomeadsorbed in perivascular regions by the first tumor cells encountered,leaving none to reach tumor cells at more distant sites (Baxter et al.,1991; Kennel et al., 1991).

[0013] Thus, it is quite clear that a significant need exists for thedevelopment of novel strategies for the treatment of solid tumors. Oneapproach involves the targeting of agents to the vasculature of thetumor, rather than to tumor cells. Solid tumor growth is highlydependent on the vascularization of the tumor and the growth of tumorcells can only be maintained if the supply of oxygen, nutrients andother growth factors and the efflux of metabolic products aresatisfactory. Indeed, it has been observed that many existing therapiesmay already have, as part of their action, a vascular-mediated mechanismof action (Denekamp, 1990).

[0014] The present inventors propose that targeting the vasculature willlikely deprive the tumor of life sustaining events and result in reducedtumor growth rate or tumor cell death. This approach is contemplated tooffer several advantages over direct targeting of tumor cells. Firstly,the target cells are directly accessible to intravenously administeredtherapeutic agents, permitting rapid localization of a high percentageof the injected dose (Kennel et al., 1991). Secondly, since eachcapillary provides oxygen and nutrients for thousands of cells in itssurrounding ‘cord’ of tumor, even limited damage to the tumorvasculature could produce an avalanche of tumor cell death (Denekamp,1990; Denekamp, 1984). Finally, the outgrowth of mutant endothelialcells, lacking a target antigen, is unlikely because they are normalcells.

[0015] At the present time, it is generally accepted that for tumorvascular targeting to succeed, antibodies are required that recognizetumor endothelial cells but not those in normal tissues. Althoughseveral antibodies have been raised (Duijvestijn et al., 1987; Hagemeieret al., 1986; Bruland et al., 1986; Murray et al., 1989; Schlingemann etal., 1985), none have shown a high degree of specificity. Also, there donot appear to be reports of any particular agents, other than theaforementioned toxins, that show promise as the second agent in avascular targeted antibody conjugate. Thus, unfortunately, whilevascular targeting presents certain theoretical advantages, effectivestrategies incorporating these advantages have yet to be developed.

SUMMARY OF THE INVENTION

[0016] The present invention overcomes the limitations of the prior artby providing novel compositions and methods for use in achievingspecific coagulation, for example, coagulation in tumor vasculature,with limiting side-effects. The invention, in a general and overallsense, concerns various novel immunological and growth factor-basedbispecific compositions capable of stimulating coagulation indisease-associated vasculature, and methods for their preparation anduse.

[0017] The invention provides binding ligands that may generally bedescribed as “bispecific binding ligands”. Such ligands comprise a“first binding region” that typically binds to a disease-related targetcell, such as a tumor cell, or to a component associated with such acell; to some component associated with disease-related vasculature,e.g., tumor vasculature; or to a component of, or associated with,disease-associated stroma. The first binding region is operativelyassociated with or linked to a “coagulating agent”, which may be eithera coagulation factor itself or may be a second binding region that iscapable of binding to a coagulation factor.

[0018] The binding ligands of the invention are described as“bispecific” as they are “at least” bispecific, i.e., they comprise, ata minimum, two functionally distinct regions. Compositions and methodsusing other constructs, such as trispecific and mutlispecific bindingligands, are also included within the scope of the invention. Combinedcompositions, kits and methods of using the bispecific coagulatingligands described herein in conjunction with other effectors, such asother immunological- and growth-factor-based compositions,antigen-inducing agents, immunostimulants, immunosuppressants,chemotherapeutic drugs, and the like, are also contemplated.

[0019] The first binding regions, and any second binding regions, may beantibodies or fragments thereof. As used herein, the term “antibody” isintended to refer broadly to any immunologic binding agent such as IgG,IgM, IgA, IgD and IgE. Generally, IgG or IgM are preferred because theyare the most common antibodies in the physiological situation andbecause they are most easily made in a laboratory setting. Monoclonalantibodies (MAbs) are recognized to have certain advantages, e.g.,reproducibility and large-scale production, and their use is generallypreferred. Engineered antibodies, such as recombinant antibodies andhumanized antibodies, also fall within the scope of the invention.

[0020] Where antigen binding regions of antibodies are employed as thebinding and targeting agent, a complete antibody molecule may beemployed. Alternatively, a functional antigen binding region may beused, as exemplified by Fv, scFv (single chain Fv), Fab′, Fab, Dab orF(ab′)₂ fragment of an antibody. The techniques for preparing and usingvarious antibody-based constructs are well known in the art and arefurther described herein.

[0021] The coagulation factor portion of the binding ligands is formedso that it maintains significant functional capacity, i.e., it is in aform so that, when delivered to the target region, it still retains itsability to promote blood coagulation or clotting. However, in certainembodiments, the coagulation factor portion of the binding ligands willbe less active than, for example, the natural counterpart of thecoagulant, and the factor will achieve the desired level of activityonly upon delivery to the target area. One such example is a vitaminK-dependent coagulation factor that lacks the Gla modification, whichwill nonetheless achieve significant functional activity upon binding ofthe first binding region of the bispecific ligand to a membraneenvironment.

[0022] Where a second binding region is used to bind a coagulationfactor, it is generally chosen so that it recognizes a site on thecoagulation factor that does not significantly impair its ability toinduce coagulation. Likewise, where a coagulation factor is covalentlylinked to a first binding agent, a site distinct from its functionalcoagulating site is generally used to join the molecules.

[0023] The “first binding region” of the bispecific ligands of theinvention may be any component that binds to a designated target site,i.e., a site associated with a tumor region or other disease site inwhich coagulation is desired. The target molecule, in the case of tumortargeting, will generally be present at a higher concentration in thetumor site than in non-tumor sites. In certain preferred embodiments,the targeted molecules, whether associated with tumor cells, tumorvascular cells, tumor-associated stroma, or other components, will berestricted to such cells or other tumor-associated entities, however,this is not a requirement of the invention.

[0024] In this regard, it should be noted that tumor vasculature is‘prothrombotic’ and is predisposed towards coagulation. It is thuscontemplated that a targeted coagulant is likely to preferentiallycoagulate tumor vasculature while not coagulating normal tissuevasculature, even if other normal cells or body components,particularly, the normal endothelial cells or even stroma, expresssignificant levels of the target molecule. This approach is thereforeenvisioned to be safer for use in humans, e.g., as a means of treatingcancer, than that of targeting a toxin to tumor vasculature.

[0025] In certain embodiments, the first binding regions contemplatedfor use in this invention may be directed to a tumor cell component orto a component associated with a tumor cell. In targeting generally to atumor cell, it is believed that the first binding ligand will cause thecoagulation factor component of the bispecific binding ligand toconcentrate on those perivascular tumor cells nearest to the bloodvessel and thus trigger coagulation of tumor blood vessels, giving thebispecific binding ligand significant utility.

[0026] A first binding region may therefore be a component, such as anantibody or other agent, that binds to a tumor cell. Agents that “bindto a tumor cell” are defined herein as ligands that bind to anyaccessible component or components of a tumor cell, or that bind to acomponent that is itself bound to, or otherwise associated with, a tumorcell, as further described herein.

[0027] The majority of such tumor-binding ligands are contemplated to beagents, particularly antibodies, that bind toga cell surface tumorantigen or marker. Many such antigens are known, as are a variety ofantibodies for use in antigen binding and tumor targeting. The inventionthus includes first binding regions, such as antigen binding regions ofantibodies, that bind to an identified tumor cell surface antigen, suchas those listed in Table I, and first binding regions thatpreferentially or specifically bind to an intact tumor cell, such asbinding to a tumor cell listed in Table II.

[0028] Currently preferred examples of tumor cell binding regions arethose that comprise an antigen binding region of an antibody that bindsto the cell surface tumor antigen p185^(HER2), milk mucin core protein,TAG-72, Lewis a or carcinoembryonic antigen (CEA). Another group ofcurrently preferred tumor cell binding regions are those that comprisean antigen binding region of an antibody that binds to atumor-associated antigen that binds to the antibody 9.2.27, OV-TL3,MOv18, B3, KS1/4, 260F9 or D612.

[0029] The antibody 9.2.27 binds to high M_(r) melanoma antigens, OV-TL3and MOv18 both bind to ovarian-associated antigens, B3 and KS1/4 bind tocarcinoma antigens, 260F9 binds to breast carcinoma and D612 binds tocolorectal carcinoma. Antigen binding moieties that bind to the sameantigen as D612, B3 or KS1/4 are particularly preferred. D612 isdescribed in U.S. Pat. No. 5,183,756, and has ATCC Accession No. HB9796; B3 is described in U.S. Pat. No. 5,242,813, and has ATCC AccessionNo. HB 10573; and recombinant and chimeric KS1/4 antibodies aredescribed in U.S. Pat. No. 4,975,369; each incorporated herein byreference.

[0030] In tumor cell targeting, where the tumor marker is a component,such as a receptor, for which a biological ligand has been identified,the ligand itself may also be employed as the targeting agent, ratherthan an antibody. Active fragments or binding regions of such ligandsmay also be employed.

[0031] First binding regions for use in the invention may also becomponents that bind to a ligand that is associated with a tumor cellmarker. For example, where the tumor antigen in question is acell-surface receptor, tumor cells in vivo will have the correspondingbiological ligand, e.g., hormone, cytokine or growth factor, bound totheir surface and available as a target. This includes both circulatingligands and “paracrine-type” ligands that may be generated by the tumorcell and then bound to the cell surface.

[0032] The present invention thus further includes first bindingregions, such as antibodies and fragments thereof, that bind to a ligandthat binds to an identified tumor cell surface antigen, such as thoselisted in Table I, or that preferentially or specifically binds to oneor more intact tumor cells. Additionally, the receptor itself, orpreferably an engineered or otherwise soluble form of the receptor orreceptor binding domain, could also be employed as the binding region ofa bispecific coagulating ligand.

[0033] In further embodiments, the first binding region may be acomponent that binds to a target molecule that is specifically orpreferentially expressed in a disease site other than a tumor site.Exemplary target molecules associated with other diseased cells include,for example, PSA associated with Benign Prostatic Hyperplasia (BPH) andFGF associated with proliferative diabetic retinopathy. It is believedthat an animal or patient having one of the above diseases would benefitfrom the specific induction of coagulation in the disease site.

[0034] This is the meaning of “diseased cell” in the present context,i.e., it is a cell that is connected with a disease or disorder, whichcell expresses, or is otherwise associated with, a targetable componentthat is present at a higher concentration in the disease sites and cellsin comparison to its levels in non-diseased sites and cells. Thisincludes targetable components that are associated with the vasculaturein the disease sites.

[0035] Exemplary first binding regions for use in targeting anddelivering a coagulant to other disease sites include antibodies, suchas anti-PSA (BPH), and GF82, GF67 3H3 that bind to FGF. Biologicalbinding ligands, such as FGF, that bind to the relevant receptor, inthis case the FGF receptor, may also be used. Antibodies againstvascular targets may also be employed, as described below. The targetingof the stroma or endothelial cells provides a powerful means of treatingother diseases where the “diseased cell” itself may not be associatedwith a strong or unique marker antigen.

[0036] In further embodiments, the first binding regions of theinvention will be components that are capable of binding to a componentof disease-associated vasculature, i.e., a region of vasculature inwhich specific coagulation would be advantageous to the animal orpatient. First binding regions capable of binding to a componentspecifically or preferentially associated with tumor vasculature arecurrently preferred. “Components of tumor vasculature” include bothtumor vasculature endothelial cell surface molecules and any components,such as growth factors, that may be bound to these cell surfacereceptors or molecules.

[0037] Certain preferred binding ligands are antibodies, and fragmentsthereof, that bind to cell surface receptors and antibodies that bind tothe corresponding biological ligands of these receptors. Exemplaryantibodies are those that bind to MHC Class II proteins, VEGF/VPFreceptors,. FGF receptors, TGFβ receptors, a TIE (tyrosinekinase-immunoglobulin-epidermal growth factor-like receptor, includingTIE-1 and TIE-2), VCAM-1, P-selectin, E-selectin, α_(v)β₃ integrin,pleiotropin, endosialin and endoglin.

[0038] First binding regions that comprise an antigen binding region ofan antibody that binds to endoglin are one group of preferred agents.These are exemplified by antibodies and fragments that bind to the sameepitope as the monoclonal antibody TEC-4 or the monoclonal antibodyTEC-11.

[0039] Antigen binding region of antibodies that bind to the VEGFreceptor are another group of preferred agents. These are particularlyexemplified by antibodies and fragments that bind to the same epitope asthe monoclonal antibody 3E11, 3E7, 5G6, 4D8, 10B10 or TEC-110. Anti-VEGFantibodies with binding specificities substantially the same as any oneof the antibodies termed 1B4, 4B7, 1B8, 2C9, 7D9, 12D2, 12D7, 12E10,5E5, 8E5, 5E11, 7E11, 3F5, 10F3, 1F4, 2F8, 2F9, 2F10, 1G6, 1G11, 3G9,9G11, 10G9, GV97, GV39, GV97γ, GV39γ, GV59 or GV14 may also be used.Further suitable anti-VEGF antibodies include 4.6.1., A3.13.1, A4.3.1and B2.6.2 (Kim et. al., 1992); SBS94.1 (Oncogene Science); G143-264 andG143-856 (Pharmingen).

[0040] Further useful antibodies are those that bind to a ligand thatbinds to a tumor vasculature cell surface receptor. Antibodies that bindto VEGF/VPF, FGF, TGFβ, a ligand that binds to a TIE, a tumor-associatedfibronectin isoform, scatter factor, hepatocyte growth factor (HGF),platelet factor 4 (PF4), PDGF (including PDGFa and PDGFb) and TIMP (atissue inhibitor of metalloproteinases, including TIMP-1, TIMP-2 andTIMP-3) are therefore useful in these embodiments, with antibodies thatbind to VEGF/VPF, FGF, TGFβ, a ligand that binds to a TIE or atumor-associated fibronectin isoform often being preferred.

[0041] In still further embodiments, it is contemplated that markersspecific for tumor vasculature may be those that have been firstinduced, i.e., their expression specifically manipulated by the hand ofman, allowing subsequent targeting using a binding ligand, such as anantibody.

[0042] Exemplary inducible antigens include those inducible by acytokine, e.g., IL-1, IL-4, TNF-α, TNF-β or IFN-γ, as may be released bymonocytes, macrophages, mast cells, helper T cells, CD8-positiveT-cells, NK cells or even tumor cells. Examples of the induced targetsare E-selectin, VCAM-1, ICAM-1, endoglin and MHC Class II antigens. Whenusing MHC Class II induction, the suppression of MHC Class II in normaltissues is generally required, as may be achieved using a cyclosporin,such as Cyclosporin A (CsA), or a functionally equivalent agent.

[0043] Further inducible antigens include those inducible by acoagulant, such as by thrombin, Factor IX/IXa, Factor X/Xa, plasmin or ametalloproteinase (matrix metalloproteinase, MMP). Generally, antigensinducible by thrombin will be used. This group of antigens includesP-selectin, E-selectin, PDGF and ICAM-1, with the induction andtargeting of P-selectin and/or E-selectin being generally preferred.

[0044] Antibodies that bind to epitopes that are present onligand-receptor complexes, but absent from both the individual ligandand receptor may also be used. Such antibodies will recognize and bindto a ligand-receptor complex, as presented at the cell surface, but willnot bind to the free ligand or uncomplexed receptor. A “ligand-receptorcomplex”, as used herein, therefore refers to the resultant complexproduced when a ligand, such as a growth factor, specifically binds toits receptor, such as a growth factor receptor. This is exemplified bythe VEGF/VEGF, receptor complex.

[0045] It is envisioned that such ligand-receptor complexes will likelybe present in a significantly higher number on tumor-associatedendothelial cells than on non-tumor associated endothelial cells, andmay thus be targeted by anti-complex antibodies. Anti-complex antibodiesinclude those antibodies and fragments thereof that bind to the sameepitope as the monoclonal antibody 2E5, 3E5 or 4E5.

[0046] In further embodiments, the first binding regions contemplatedfor use in this invention will bind to a component of disease-associatedstroma, such as a component of tumor-associated stroma. This includesantigen binding regions of antibodies that bind to basement membranecomponents, activated platelets and inducible tumor stroma components,especially those inducible by a coagulant, such as thrombin. “Activatedplatelets” are herein defined as a component of tumor stroma, one reasonfor which being that they bind to the stroma when activated.

[0047] Preferred targetable elements of tumor-associated stroma arecurrently the tumor-associated fibronectin isoforms,. Fibronectinisoforms are ligands that bind to the integrin family of receptors.Tumor-associated fibronectin isoforms are available, e.g., as recognizedby the MAb BC-1. This Mab, and others of similar specificity, aretherefore preferred agents for use in the present invention. Fibronectinisoforms, although stromal components, bind to endothelial cells and maythus be considered as a targetable vascular endothelial cell-boundligand in the context of the invention.

[0048] Another group of preferred anti-stromal antibodies are those thatbind to RIBS, the receptor-induced binding site, on fibrinogen. RIBS isa targetable antigen, the expression of which in stroma is dictated byactivated platelets. Antibodies that bind to LIBS, the ligand-inducedbinding site, on activated platelets are also useful.

[0049] One further group of useful antibodies are those that bind totenascin, a large molecular weight extracellular glycoprotein expressedin the stroma of various benign and malignant tumors. Antibodies such asthose described by Shrestha et. al. (1994) and 143DB7C8, described byTuominen & Kallioinen (1994), may thus be used as the binding portionsof the coaguligands.

[0050] “Components of disease- and tumor-associated stroma” includevarious cell types, matrix components, effectors and other moleculescomponents that may be considered, by some, to be outside the narrowestdefinition of “stroma”, but are nevertheless targetable entities thatare preferentially associated with a disease region, such as a tumor.

[0051] Accordingly, the first binding region may be an antibody orligand that binds to a smooth muscle cell, a pericyte, a fibroblast, amacrophage, an infiltrating lymphocyte or leucocyte. First bindingregions may also bind to components of the connective tissue, andinclude antibodies and ligands that bind to, e.g., fibrin,proteoglycans, glycoproteins, collagens, and anionic polysaccharidessuch as heparin and heparin-like compounds.

[0052] In other preferred embodiments, the vasculature and stromabinding ligands of the invention will be binding regions that arethemselves biological ligands, or portions thereof, rather than anantibodies. “Biological ligands” in this sense will be those moleculesthat bind to or associate with cell surface molecules, such asreceptors, that are accessible in the stroma or on vascular cells; asexemplified by cytokines, hormones, growth factors, and the like. Anysuch growth factor or ligand may be used so long as it binds to thedisease-associated stroma or vasculature, e.g., to a specific biologicalreceptor present on the surface of a tumor vasculature endothelial cell.

[0053] Suitable growth factors for use in these aspects of the inventioninclude, for example, VEGF/VPF (vascular endothelial growthfactor/vascular permeability factor), FGF (the fibroblast growth factorfamily of proteins), TGFβ (transforming growth factor B), a ligand thatbinds to a TIE, a tumor-associated fibronectin isoform, scatter factor,hepatocyte growth factor (HGF), platelet factor 4 (PF4), PDGF (plateletderived growth factor), TIMP or even IL-8, IL-6 or Factor XIIIa.VEGF/VPF and FGF will often be preferred.

[0054] Targeting an endothelial cell-bound component, e.g., a cytokineor growth factor, with a binding ligand construct based on a knownreceptor is also contemplated. Generally, where a receptor is used as atargeting component, a truncated or soluble form of the receptor will beemployed. In such embodiments, it is particularly preferred that thetargeted endothelial cell-bound component be a dimeric ligand, such asVEGF. This is preferred as one component of the dimer will already bebound to the cell surface receptor in situ, leaving the other componentof the dimer available for binding the soluble receptor portion of thebispecific coagulating ligand.

[0055] The use of bispecific, or tri- or multi-specific, ligands thatinclude at least one targeting region capable of binding to a componentof disease-associated vasculature has the advantage that vascularendothelial cells, and disease-associated agents such as activatedplatelets, are similar in different diseases, and particularly indifferent tumors. This phenomenon makes it feasible to treat numerousdiseases and types of cancer with one pharmaceutical, rather than havingto tailor the agent to each individual disease or specific tumor type.

[0056] The compositions and methods of the present invention are thussuitable for use in treating both benign and malignant diseases thathave a vascular component. Such vasculature-associated diseases includebenign growths, such as BPH, diabetic retinopathy, vascular restenosis,arteriovenous malformations (AVM), meningioma, hemangioma, neovascularglaucoma and psoriasis. Also included within this group are synovitis,dermatitis, endometriosis, angiofibroma, rheumatoid arthritis,atherosclerotic plaques, corneal graft neovascularization, hemophilicjoints, hypertrophic scars, osler-weber syndrome, pyogenic granulomaretrolental fibroplasia, scleroderma, trachoma, and vascular adhesions.Each of the above diseases are known to have a common angio-dependentpathology, it is thus contemplated that achieving coagulation in thedisease site would prove beneficial.

[0057] The bispecific binding ligand-coagulation factor conjugates ofthe present invention may be conjugates in which the two or morecomponents are covalently linked. For example, by using a biochemicalcrosslinker and, preferably, one that has reasonable stability in blood,as exemplified by SMPT. The components may also be linked using thewell-known avidin (or streptavidin) and biotin combination. Variouscross-linkers, avidin:biotin compositions and combinations, andtechniques for preparing conjugates, are well known in the art and arefurther described herein.

[0058] Alternatively, such bispecific coagulating agents may be fusionproteins prepared by molecular biological techniques, i.e., by joining agene (or cDNA) encoding a binding ligand or region to a gene (or cDNA)encoding a coagulation factor. This is well known in the art and isfurther described herein. Typically, an expression vector is preparedthat comprises, in the same reading frame, a DNA segment encoding thefirst binding region operatively linked to a DNA segment encoding thecoagulation factor and expressing the vector in a recombinant host cellso that it produces the encoded fusion protein.

[0059] Coagulation factors for use in the invention may comprise one ofthe vitamin K-dependent coagulant factors, such as Factor II/IIa, FactorVII/VIIa, Factor IX/IXa or Factor X/Xa. Factor V/Va, VIII/VIIIa, FactorXI/XIa, Factor XII/XIIa and Factor XIII/XIIIa may also be used.

[0060] Particular aspects concern the vitamin K-dependent coagulationfactors that lack the Gla modification. Such factors may be prepared byexpressing a vitamin K-dependent coagulation factor-encoding gene in aprocaryotic host cell (which cells are unable to effect the Glu to Glamodification). The factors may also be prepared by making an engineeredcoagulation factor gene that encodes a vitamin K-dependent coagulationfactor lacking the necessary or “corresponding” Glutamic acid residues,and then expressing the engineered gene in virtually any recombinanthost cell. Equally, such a coagulation factor may be prepared bytreating the vitamin K-dependent coagulation factor protein to remove oralter the corresponding Glutamic acid residues.

[0061] Preferred coagulation factors for use in the binding ligands ofthe invention are Tissue Factor and Tissue Factor derivatives. One groupof useful Tissue Factors are those mutants deficient in the ability toactivate Factor VII. A Tissue Factor may be rendered deficient in theability to activate Factor VII by altering one or more amino acids fromthe region generally between about position 157 and about position 167in the amino acid sequence. Exemplary mutants are those wherein Trp atposition 158 is changed to Arg; wherein Ser at position 162 is changedto Ala; wherein Gly at position 164 is changed to Ala; and the doublemutant wherein Trp at position 158 is changed to Arg and Ser at position162 is changed to Ala.

[0062] Further preferred Tissue Factor derivatives are truncated TissueFactors, dimeric or even polymeric Tissue Factors and dimeric, or evenpolymeric, truncated Tissue Factors.

[0063] The present invention further provides novel Tissue Factorconstructs that comprise a Tissue Factor or derivative operativelylinked to at least one other Tissue Factor or derivative. TruncatedTissue Factors are preferred, with truncated Tissue Factors that havebeen modified to comprise a hydrophobic membrane insertion moiety beingparticularly preferred.

[0064] “A hydrophobic membrane insertion moiety”, as defined herein, isone or more units that direct the insertion or functional contact of theTissue Factor with a membrane. The hydrophobic membrane insertionmoieties of the invention are exemplified by stretches of substantiallyhydrophobic amino acids, such as between about 3 and about 20hydrophobic amino acids; and also by fatty acids.

[0065] The hydrophobic amino acids may be located either at the N- orC-terminus of the truncated Tissue Factor, or appended at another pointof the molecule. Where hydrophobic amino acids are used, they may beadvantageously incorporated into the molecule by molecular biologicaltechniques. Equally, hydrophobic amino acids or fatty acids may be addedto the Tissue Factor using synthetic chemistry techniques.

[0066] In the Tissue Factor dimers, trimers and polymers of the presentinvention, each of the Tissue Factors or derivatives may be operativelylinked via, e.g., a disulfide, thioether or peptide bond. In certainembodiments, the Tissue Factor units will be linked via a bond that issubstantially stable in plasma, or in the physiological environment inwhich it is intended for use. This is based upon the inventors' conceptthat the dimeric form of Tissue Factor may prove to be the mostbiologically active. However, there is no requirement for a stablelinkage as Tissue Factor monomers are known to be active in the methodsof the invention.

[0067] One or more of the Tissue Factors or truncated Tissue Factors inthe dimers and multimers may also be modified to contain a terminalcysteine residue or another moiety that is suitable for linking theTissue Factor construct to a second agent, such as a binding region.

[0068] Tissue Factor monomers, truncated Tissue Factors, and TissueFactor dimers and multimers that contain a peptide that includes aselectively-cleavable amino acid sequence therefore form another aspectof the invention. Peptide linkers that include a cleavage site forurokinase, plasmin, Thrombin, Factor IXa, Factor Xa or ametalloproteinase, such as an interstitial collagenase, a gelatinase ora stromelysin, are particularly preferred.

[0069] The Tissue Factor monomers, truncated Tissue Factors, TissueFactor dimers and multimers, and indeed any coagulant, may therefore belinked to a second agent, such as an antibody, an antigen binding regionof an antibody, a ligand or a receptor, via a biologically-releasablebond. The preference for peptide linkers that include a cleavage sitefor the above listed proteinases is based on the presence of suchproteinases within, e.g., a tumor environment. The delivery of abispecific agent or ligand to the tumor site is expected to result incleavage, resulting in the relatively specific release of thecoagulation factor.

[0070] Particular constructs of the invention are those comprising anoperatively linked series of units in the sequence: a cysteine residue,a selectively cleavable peptide linker, a stretch of hydrophobic aminoacids, a first truncated Tissue Factor and a second truncated TissueFactor; or in the sequence: a first cysteine residue, a selectivelycleavable peptide linker, a first stretch of hydrophobic amino acids, afirst truncated Tissue Factor, a second truncated Tissue Factor and asecond stretch of hydrophobic amino acids; wherein each construct may ornot be linked to a second agent such as an antibody, ligand or receptor.

[0071] Other suitable coagulation factors are Russell's viper venomFactor X activator; platelet-activating compounds, such as thromboxaneA₂ and thromboxane A₂ synthase; and inhibitors of fibrinolysis, such asα2-antiplasmin.

[0072] Also encompassed by the invention are binding ligands in whichthe coagulation factor is not covalently linked to the conjugate, but isnon-covalently bound thereto by means of binding to a second bindingregion that is operatively linked to the targeting agent of theconstruct. Suitable “second binding regions” include antigen combiningsites of antibodies that have binding specificity for the coagulationfactor, including functional portions of antibodies, such as scFv, Fv,Fab′, Fab and F(ab′)₂ fragments.

[0073] Binding ligands that contain antibodies, or fragments thereof,directed against the vitamin K-dependent coagulant Factor II/IIa, FactorVII/VIIa, Factor IX/IXa or Factor X/Xa; a vitamin K-dependentcoagulation factor that lacks the Gla modification; Tissue Factor, amutant Tissue Factor, a truncated Tissue Factor, a dimeric TissueFactor, a polymeric Tissue Factor, a dimeric truncated Tissue Factor;Prekallikein; Factor V/Va, VIII/VIIIa, Factor XI/XIa, Factor XII/XIIa,Factor XIII/XIIIa; Russell's viper venom Factor X activator, thromboxaneA₂ or α2-antiplasmin are therefore contemplated.

[0074] The non-covalently bound coagulating agents may be bound to, or“precomplexed”, with a coagulation factor, e.g., so that they may beused to deliver an exogenous coagulation factor to a disease site, e.g.,the tumor vasculature, of an animal upon administration. Equally,binding ligands that comprise a second binding region that is specificfor a coagulation factor may also be administered to an animal in an“uncomplexed” form and still function to achieve specific coagulation;in which instance, the agent would garner circulating (endogenous)coagulation factor and concentrate it within the disease or tumor site.

[0075] In terms of the “coagulation factors” or coagulating agents,these may be endogenous coagulation factors and derivatives thereof, orexogenously added version of such factors, including recombinantversions. Coagulants (in the present “coaguligands”) have the distinctadvantage over toxins (in immunotoxins) as they will not producesignificant adverse side effects upon targeting to a marker that provesto be less than 100% disease-restricted. Furthermore, the coagulantsused will most often be of human origin, and will therefore pose lessimmunogenicity problems than foreign toxins, such as ricin A chain.

[0076] Although not limited to such compositions, important examples ofcompositions in accordance with this invention are bispecificantibodies, which antibodies comprise a first antigen binding regionthat binds to a disease cell or component of disease-associatedvasculature marker and a second antigen binding region that binds to acoagulation factor. The invention also provides scfv, Fv, Fab′, Fab andF(ab′)₂ fragments of such bispecific antibodies. One currently preferredexample of such a bispecific antibody is an antibody comprising onebinding site directed against an MHC Class II antigen and anotherbinding site directed against Tissue Factor.

[0077] In further embodiments, the present invention providespharmaceutical compositions of, and therapeutic kits comprising, any ora combination of the above binding ligands and bispecific antibodies inpharmacologically acceptable forms. This includes pharmaceuticalcompositions and kits where the binding ligand has a first bindingregion that is covalently linked to a coagulation factor, and alsobinding ligands in which the first binding region is covalently linkedto a second binding region that, in turn, binds to the coagulationfactor—whether binding occurs prior to, or subsequent to, administrationto an animal.

[0078] Pharmaceutical compositions and therapeutic kits that include acombination of bispecific, trispecific or multispecific binding ligandsin accordance with the invention are also contemplated. This includescombinations where one binding ligand is directed against a diseasedcell or a tumor cell and where another is directed against a vasculatureendothelial cell marker or component of disease-associated stroma. Otherdistinct components may also be included in the compositions and kits ofthe invention, such as antibodies, immunotoxins, immunoeffectors,chemotherapeutic agents, and the like.

[0079] The kits may also include an antigen suppressor, such as acyclosporin, for use in suppressing antigen expression in endothelialcells of normal tissues; and/or an “inducing agent” for use in inducingdisease-associated vascular endothelial cells or stroma to express atargetable antigen, such as E-selectin, E-selectin or an MHC Class IIantigen. Exemplary inducing agents include T cell clones that binddisease or tumor antigens and that produce IFN-γ, although it iscurrently preferred that such clones be isolated from the animal to betreated using the kit.

[0080] Preferred inducing agents are bispecific antibodies that bind todisease or tumor cell antigens, or even stromal components, and toeffector cells capable of producing cytokines, coagulants, or otherfactors, that induce expression of desired target antigens. Currently,one preferred group of bispecific antibodies are those that bind to atumor antigen and to the activation antigens CD14 or CD16, to stimulateIL-1 production by monocytes, macrophages or mast cells; and those thatbind to a tumor antigen and to the activation antigens CD2, CD3 or CD28,and preferably CD28, to stimulate IFN-γ production by NK cells orpreferably by T cells.

[0081] A second preferred group of bispecific antibodies are those thatbind to a tumor antigen or to a component of tumor stroma, and to TissueFactor, a Tissue Factor derivative, prothrombin, Factor VII/VIIa, FactorIX/IXa, Factor X/Xa, Factor XI/XIa or Russell's viper venom Factor Xactivator, to stimulate thrombin production. Kits comprising suchbispecific antibodies as a first “inducing” composition will generallyinclude a second pharmaceutical composition that comprises a bindingligand that comprises a first binding region that binds to P-selectin orE-selectin.

[0082] The bispecific ligands of the invention, and other components asdesired, may be conveniently aliquoted and packaged, using one or moresuitable container means, and the separate containers dispensed in asingle package. Pharmaceutical compositions and kits are furtherdescribed herein.

[0083] Although the present invention has significant clinical utilityin the delivery of coagulants and in disease treatment, it also has manyin vitro uses. These include, for example, various assays based upon thebinding ability of the particular antibody, ligand or receptor, of thebispecific compounds. The bispecific coagulating ligands of inventionmay thus be employed in standard binding assays and protocols, such asin immunoblots, Western blots, dot blots, RIAs, ELISAs,immunohistochemistry, fluorescent activated cell sorting (FACS),immunoprecipitation, affinity chromatography, and the like, as furtherdescribed herein.

[0084] In still further embodiments, the invention concerns methods fordelivering a coagulant to disease-associated vasculature, as may be usedto treat diseases such as diabetic retinopathy, vascular restenosis,AVM, hemangioma, neovascular glaucoma, psoriasis and rheumatoidarthritis, and tumors that have a vascularized tumor component. Suchmethods generally comprise administering to an animal, including a humansubject, with a disease that has a vascular component, a pharmaceuticalcomposition comprising at least one bispecific binding ligand inaccordance with those described above.

[0085] The compositions are administered in amounts and by routeseffective to promote blood coagulation in the vasculature of the diseasesite, e.g., a solid tumor. Effective doses will be known to those ofskill in the art in light of the present disclosure, such as theinformation in the Preferred Embodiments and Detailed Examples.Parenteral administration will often be suitable, as will other methods,such as, e.g., injection into a vascularized tumor site.

[0086] The methods of the invention provide for the delivery ofexogenous coagulation factors, by means of both administering a bindingligand that comprises a covalently-bound coagulation factor and by meansof administering a binding ligand that comprises a non-covalently boundcoagulation factor that is complexed to a second binding region of thebispecific ligand or antibody.

[0087] Further methods of the invention include those that result in thedelivery of an endogenous coagulation factor to disease or tumorvasculature. This is achieved by administering to the animal or patienta binding ligand that comprises a second binding region that binds toendogenous coagulation factor and concentrates the factor at thedisease-associated or tumor vasculature.

[0088] In yet still further methodological embodiments, it iscontemplated that markers of tumor vasculature or stroma may bespecifically induced and then targeted using a binding ligand, such asan antibody. Exemplary inducible antigens include E-selectin,P-selectin, MHC Class II antigens, VCAM-1, ICAM-1, endoglin, ligandsreactive with LAM-1, vascular addressins and other adhesion molecules,with E-selectin and MHC Class II antigens being currently preferred.

[0089] When inducing and subsequently targeting MHC Class II proteins,the suppression of MHC Class II in normal tissues is generally required.MHC Class II suppression may be achieved using a cyclosporin, or afunctionally equivalent agent. MHC Class II molecules may then beinduced in disease-associated vascular endothelial cells usingcyclosporin-independent means, such as by exposing thedisease-associated vasculature to an effector cell, generally a Helper Tcell or NK cell, of the animal that releases the inducing cytokineIFN-γ.

[0090] Activated monocytes, macrophages and even mast cells are effectorcells capable of producing cytokines (IL-1; TNF-α; TNF-β) that induceE-selectin; whereas Helper T cells, CD8-positive T cells and NK cellsare capable of producing IFN-γ that induces MHC Class II. Activatingmonocyte/macrophages in the disease site to produce IL-1, or activatingdisease-associated Helper T cells or NK cells to produce IFN-γ, may beachieved by administering to the animal an activating antibody thatbinds to an effector cell surface activating antigen. Exemplaryactivating antigens include CD14 and CD16 (FcR for IgE) formonocytes/macrophages; and CD2, CD3 and CD28 for T cells; with CD14 andCD28, respectively, being preferred for use in certain embodiments.

[0091] To achieve specific activation and induction, one currentlypreferred method is to use a bispecific antibody that binds to both aneffector cell activating antigen, such as CD14 or CD28, and to a diseaseor tumor cell antigen. These bispecific antibodies will localize to thedisease or tumor site and activate monocyte/macrophages and T cells,respectively. The activated effector cells in the vicinity of thetargeted disease or tumor component will produce inducing cytokines, inthis case, IL-1 and IFN-γ, respectively.

[0092] MHC Class II suppression in normal tissues may also be achievedby administering to an animal an anti-CD4 antibody; this functions tosuppress IFN-γ production by T cells of the animal resulting ininhibition of MHC Class II expression. MHC Class II molecules may againbe specifically induced in disease-associated vascular endothelial cellsby exposing only the disease site to IFN-γ. One means by which toachieve this is by administering to the animal an IFN-γ-producing T cellclone that binds to an antigen in the disease site. The IFN-γ-producingT cells will preferably be infiltrating leukocytes obtained from thedisease site of the animal, such as tumor infiltrating leukocytes (TILs)expanded in vitro.

[0093] Method using bispecific antibodies to induce coagulant, such asthrombin, production, only in a local environment, such as in a tumorsite, are also provided. Again, this will generally be achieved byadministering to an animal a pharmaceutical composition comprising abispecific antibody that binds to a tumor cell or a component of tumorstroma and to Tissue Factor, a Tissue Factor derivative, prothrombin,Factor VII/VIIa, Factor IX/IXa, Factor X/Xa, Factor XI/XIa or Russell'sviper venom Factor X activator. Antibodies that bind to E-selectin orP-selectin are then linked to a coagulation factor or a second bindingregion that binds to a coagulation factor and are introduced into thebloodstream of an animal.

[0094] More conventional combination treatment regimens are alsopossible where, for example, a tumor coagulating element of thisinvention is combined with an existing antitumor therapy, such as withradiotherapy or chemotherapy, or through the use of a secondimmunological reagent, such as an antitumor immunotoxin. The noveltreatment methods for benign diseases can also be combined with otherpresently used therapies.

BRIEF DESCRIPTION OF THE DRAWINGS

[0095] The following drawings form part of the present specification andare included to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

[0096]FIG. 1. Tethering of tTF to A20 cells via B21-2/10H10 bispecificantibody. A20 cells were incubated with varying concentrations ofB21-2/10H10 (□), SFR8/10H10 () or B21-2/OX7 (◯) plus an excess of¹²⁵I-tTF for 1 h at 4° C. in the presence of sodium azide. The number of¹²⁵I-tTF associated with the cells was determined as described inExample II.

[0097]FIG. 2. Relationship between number of tethered tTF molecules perA20 cell and ability to induce coagulation of plasma. A20 cells wereincubated with varying concentrations of B21-2/10H10 plus an excess oftTF for 1 h at 4° C. in the presence of sodium azide. The cells werewashed, warmed to 37° C., calcium and mouse plasma were added and thetime for the first fibrin strands to form was recorded (abscissa). Anidentical study was performed in which the A20 cells were incubated for1 h at 4° C. with bispecific antibody plus ¹²⁵I-tTF and the number oftTF specifically bound to the cells was determined as described inExample II (ordinate). Plasma added to untreated A20 cells (i.e. zerotTF molecules/cell) coagulated in 190 seconds.

[0098]FIG. 3A, FIG. 3B, FIG. 3C and FIG. 3D. Time course of vascularthrombosis and tumor necrosis after administration of coaguligand.Groups of 3 mice bearing 0.8 cm diameter C1300 (Muγ) tumors were givenan intravenous injection of a coaguligand composed of 14 μg B21-2/10H10and 11 μg tTF. FIG. 3A; Before injection: blood vessels are intact andtumor cells are healthy. FIG. 3B; 0.5 hours: blood vessels throughoutthe tumor are thrombosed; tumor cells are healthy. FIG. 3C; 4 hours:dense thrombi are present in all tumor vessels and tumor cells areseparating and developing pyknotic nuclei. Erythrocytes are visible inthe tumor interstitium. FIG. 3D; 24 hours: advanced tumor necrosisthroughout the tumor. Arrows indicate blood vessels.

[0099]FIG. 4. Solid tumor regression induced by tumor-vasculaturedirected coaguligand therapy. Nu/nu mice bearing approximately 0.8 cmdiameter C1300 (Muγ) tumors were given two intravenous injections ofB21-2/10H10 (14 μg) mixed with tTF (11 μg) spaced 1 week apart (arrows)(□). Mice in control groups received equivalent doses of tTF alone (),B21-2/10H10 alone (◯) or diluent (▪). Other control groups whichreceived equivalent doses of isotype-matched control bispecificantibodies (SFR8/10H10, OX7/10H10 or B21-2/OX7) and tTF had similartumor responses to those in animals receiving tTF alone. The number ofmice per group was 7 or 8.

[0100]FIG. 5. Exemplary antibody-tTF constructs. This figure shows boththe conjugates synthesized by the linkage of chemically derivatizedantibody to chemically derivatized tTF via a disulfide bond, and alsothe linkage of various TF or TF dimers to antibodies and fragmentsthereof.

[0101]FIG. 6. Clotting activity of tTF conjugates when bound to A20cells. A20 cells were incubated with varying concentrations ofB21-2/10H10 bispecific+H₆[tTF] in a 1:1 molar ratio, premixed for onehour (□), B21-2 antibody-H₆ C[tTF] (), and B21-2 antibody-H₆[tTF] (▴)for 1 hour at 4° C. in the presence of sodium azide. The cells werewashed, warmed to 37° C., calcium and mouse plasma were added and thetime for the first fibrin strands to form was recorded. The results areexpressed as clotting time as a % of the clotting time in the absence oftTF.

[0102]FIG. 7. Clotting activity of anti-tumor cell tTF conjugates.LS174T cells (▪), Widr cells () and H460 cells (▴), preincubated withTF9-6B4 and TF8-5G9 antibodies, were incubated with varyingconcentrations of D612 antibody-H₆C[tTF] (▪), KS1/4 antibody-H₆[tTF](), and XMMCO791 antibody-H₆[tTF] (▴) for 1 hour at 4° C. in thepresence of sodium azide. The cells were washed, warmed to 37° C.,calcium and mouse plasma were added and the time for the first fibrinstrands to form was recorded. The results are expressed as clotting timeas a % of the clotting time in the absence of tTF.

[0103]FIG. 8. Gla domains (γ-carboxyglutamic acid) of Factor II/IIa,Factor VII/IIa, Factor IX/IXa and Factor X/Xa. The arrows representsignal peptide and pro-peptide cleavage sites and activating cleavagesites (slanted arrows).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0104] Although they show great promise in the therapy of lymphomas andleukemias (Lowder et al., 1987; Vitetta et al., 1991), monoclonalantibodies (MAbs) and immunotoxins (ITs) have thus far proved relativelyineffective in clinical trials against carcinomas and other solid tumors(Byers & Baldwin, 1988; Abrams & Oldham, 1985), which account for morethan 90% of all cancers in man (Shockley et al., 1991). A principalreason for this is that macromolecules do not readily extravasate intosolid tumors (Sands, 1988; Epenetos et al., 1986) and, once within thetumor mass, fail to distribute evenly due to the presence of tightjunctions between tumor cells (Dvorak et al., 1991), fibrous stroma(Baxter et al., 1991), interstitial pressure gradients (Jain, 1990) andbinding site barriers (Juweid et al., 1992).

[0105] In developing new strategies for treating solid tumors, themethods that involve targeting the vasculature of the tumor, rather thanthe tumor cells themselves, therefore seem to offer certain advantages.Inducing a blockade of the blood flow through the tumor, e.g., throughtumor vasculature specific fibrin formation, would interfere with theinflux and efflux processes in a tumor site, thus resulting inanti-tumor effect. Arresting the blood supply to a tumor may beaccomplished through shifting the procoagulant-fibrinolytic balance inthe tumor-associated vessels in favour of the coagulating processes byspecific exposure to coagulating agents.

[0106] The present invention provides various means for effectingspecific blood coagulation, as exemplified by tumor-specificcoagulation. This is achieved using bispecific or mutlispecific bindingligands in which at least one component is an immunological- or growthfactor-based targeting component, and at least one other component isprovided that is capable of directly, or indirectly, stimulatingcoagulation.

[0107] A. Targetable Disease Sites

[0108] The compositions and methods provided by this invention arebroadly applicable to the treatment of any disease, such as a benign ormalignant tumor, having a vascular component. Suchvasculature-associated diseases include BPH, diabetic retinopathy,vascular restenosis, arteriovenous malformations (AVM), meningioma,hemangioma, neovascular glaucoma and psoriasis; and also angiofibroma,arthritis, atherosclerotic plaques, corneal graft neovascularization,hemophilic joints, hypertrophic scars, osler-weber syndrome, pyogenicgranuloma retrolental fibroplasia, scleroderma, trachoma, vascularadhesions, synovitis, dermatitis and even endometriosis.

[0109] Typical vascularized tumors are the solid tumors, particularlycarcinomas, which require a vascular component for the provision ofoxygen and nutrients. Exemplary solid tumors that may be treated usingthe invention include, but are not limited to, carcinomas of the lung,breast, ovary, stomach, pancreas, larynx, esophagus, testes, liver,parotid, biliary tract, colon, rectum, cervix, uterus, endometrium,kidney, bladder, prostate, thyroid, squamous cell carcinomas,adenocarcinomas, small cell carcinomas, melanomas, gliomas,neuroblastomas, and the like.

[0110] One binding region of the bispecific agents of the invention willbe a component that is capable of delivering the coagulating agent tothe tumor region, i.e., capable of localizing within a tumor site, suchas those described above. As somewhat wider distribution of thecoagulating agent will not be associated with severe side effects, suchas is known to occur with a toxin moiety, there is a less stringentrequirement imposed on the targeting element of the bispecific ligand.The targeting agent may thus be directed to components of tumor cells;components of tumor vasculature; components that bind to, or aregenerally associated with, tumor cells; components that bind to, or aregenerally associated with, tumor vasculature; components of the tumorextracellular matrix or stroma; and even cell types found within thetumor vasculature.

[0111] The burden of very stringent targeting, e.g., as imposed whenusing immunotoxins, is also lessened due to the fact that tumorvasculature is ‘prothrombotic’ and is predisposed towards coagulation.Therefore, to achieve specific targeting means that coagulation ispromoted in the tumor vasculature relative to the vasculature innon-tumor sites. Thus, specific targeting is a functional term ratherthan a purely physical term relating to the biodistribution propertiesof the targeting agent, and it is not unlikely that useful targets maybe not be entirely tumor-restricted, and that targeting ligands whichare effective to promote tumor-specific coagulation may nevertheless befound at other sites of the body following administration.

[0112] 1. Tumor Cell Targets

[0113] The malignant cells that make up the tumor may be targeted usinga bispecific ligand that has a region capable of binding to a relativelyspecific marker of the tumor cell. In that binding to tumor cells willlocalize the associated coagulating agent to the tumor, specificcoagulation will be achieved. Furthermore, it is expected that thiswould be a particularly effective means of promoting coagulation as, dueto the physical accessibility of perivascular tumor cells, thebispecific agents will likely be concentrated around the tumor cellsthat are nearest to a blood vessel.

[0114] Many so-called “tumor antigens” have been described, any onewhich could be employed as a target in connection with the presentinvention. A large number of exemplary solid tumor-associated antigensare listed herein in Table I. The preparation and use of antibodiesagainst such antigens is well within the skill of the art, and exemplaryantibodies are also listed in Table I.

[0115] Another means of defining a targetable tumor is in terms of thecharacteristics of a tumor cell itself, rather than describing thebiochemical properties of an antigen expressed by the cell. Accordingly,Table II is provided for the purpose of exemplifying human tumor celllines that are publically available (from ATCC Catalogue).

[0116] The information presented in Table II is by means of an example,and not intended to be limiting either by year or by scope. One mayconsult the ATCC Catalogue of any subsequent year to identify otherappropriate cell lines. Also, if a particular cell type is desired, themeans for obtaining such cells, and/or their instantly available source,will be known to those of skill in the particular art. An analysis ofthe scientific literature will thus readily reveal an appropriate choiceof cell for any tumor cell type desired to be targeted. TABLE I MARKERANTIGENS OF SOLID TUMORS AND CORRESPONDING MONOCLONAL ANTIBODIES AntigenIdentity/ Monoclonal Tumor Site Characteristics Antibodies Reference A:Gynecological ‘CA 125’ > 200 OC 125 Kabawat et al., 1983; Szymendera,1986 GY kD mucin GP ovarian 80 Kd GP OC 133 Masuko et al, Cancer Res.,1984 ovarian ‘SGA’ 360 Kd GP OMI de Krester et al., 1986 ovarian HighM_(r) mucin Mo v1 Miotti et al, Cancer Res., 1985 ovarian High M_(r)mucin/ Mo v2 Miotti et al, Cancer Res., 1985 glycolipid ovarian NS 3C2Tsuji et al., Cancer Res., 1985 ovarian NS 4C7 Tsuji et al., CancerRes., 1985 ovarian High M_(r) mucin ID₃ Gangopadhyay et al., 1985ovarian High M_(r) mucin DU-PAN-2 Lan et al., 1985 GY 7700 Kd GP F 36/22Croghan et al., 1984 ovarian ‘gp 68’ 48 Kd 4F₇/7A₁₀ Bhattacharya et al.,1984 GP GY 40, 42 kD GP OV-TL3 Poels et al., 1986 GY ‘TAG-72’ High B72.3Thor et al., 1986 M_(r) mucin ovarian 300-400 Kd GP DF₃ Kufe et al.,1984 ovarian 60 Kd GP 2C₈/2F₇ Bhattacharya et al., 1985 GY 105 Kd GP MF116 Mattes et al., 1984 ovarian 38-40 kD GP MOv18 Miotti et al., 1987 GY‘CEA’ 180 Kd GP CEA 11-H5 Wagener et al., 1984 ovarian CA 19-9 or GICACA 19-9 (1116NS 19-9) Atkinson et al., 1982 ovarian ‘PLAP’ 67 Kd GPH17-E2 McDicken et al., 1985 ovarian 72 Kd 791T/36 Perkins et al., 1985ovarian 69 Kd PLAP NDOG₂ Sunderland et al., 1984 ovarian unknown M_(r)PLAP H317 Johnson et al., 1981 ovarian p185^(HER2) 4D5, 3H4, 7C2, 6E9,Shepard et al., 1991 2C4, 7F3, 2H11, 3E8, 5B8, 7D3, SB8 uterus ovaryHMFG-2 HMFG2 Epenetos et al., 1982 GY HMFG-2 3.14.A3 Burchell et al.,1983 B: BREAST 330-450 Kd GP DF3 Hayes et al., 1985 NS NCRC-11 Ellis etal., 1984 37 kD 3C6F9 Mandeville et al., 1987 NS MBE6 Teramoto et al.,1982 NS CLNH5 Glassy et al., 1983 47 Kd GP MAC 40/43 Kjeldsen et al.,1986 High M_(r) GP EMA Sloane et al., 1981 High M_(r) GP HMFG1 HFMG2Arklie et al., 1981 NS 3.15.C3 Arklie et al., 1981 NS M3, M8, M24 Fosteret al., 1982 1 (Ma) blood M18 Foster et al., 1984 group Ags NS 67-D-11Rasmussen et al., 1982 oestrogen D547Sp, D75P3, H222 Kinsel et al., 1989receptor EGF Receptor Anti-EGF Sainsbury et al., 1985 Laminin LR-3 HoranHand et al., 1985 Receptor erb B-2 p185 TA1 Gusterson et al., 1988 NSH59 Hendler et al., 1981 126 Kd GP 10-3D-2 Soule et al., 1983 NS HmABl,2 Imam et al., 1984; Schlom et al., 1985 NS MBR 1, 2, 3 Menard et al.,1983 95 Kd 24.17.1 Thompson et al., 1983 100 Kd 24.17.2 (3E1.2) Croghanet al., 1983 NS F36/22.M7/105 Croghan et al., 1984 24 Kd C11, G3, H7Adams et al., 1983 90 Kd GP B6.2 Colcher et al., 1981 CEA & 180 Kd GPB1.1 Colcher et al., 1983 colonic & Cam 17.1 Imperial Cancer ResearchTechnology MAb pancreatic listing mucin similar to Ca 19-9 milk mucincore SM3 Imperial Cancer Research Technology Mab protein listing milkmucin core SM4 Imperial Cancer Research Technology Mab protein listingaffinity- C-Mul (566) Imperial Cancer Research Technology Mab purifiedmilk listing mucin p185^(HER2) 4D5 3H4, 7C2, 6E9, Shepard et al., 19912C4, 7F3, 2H11, 3E8, 5B8, 7D3, 5B8 CA 125 > 200 Kd OC 125 Kabawat etal., 1985 GP High M_(r) mucin/ MO v2 Miotti et al., 1985 glycolipid HighM_(r) mucin DU-PAN-2 Lan et al., 1984 ‘gp48’ 48 Kd GP 4F₇/7A₁₀Bhattacharya et al., 1984 300-400 Kd GP DF₃ Kufe et al., 1984 ‘TAG-72’high B72.3 Thor et al., 1986 M_(r) mucin ‘CEA’ 180 Kd GP cccccCEA 11Wagener et al., 1984 ‘PLAP’ 67 Kd GP H17-E2 McDicken et al., 1985HMFG-2 > 400 Kd 3.14.A3 Burchell et al., 1983 GP NS FO23C5 Riva et al.,1988 C: COLORECTAL TAG-72 High M_(r) B72.3 Colcher et al., 1987 mucinGP37 (17-1A) 1083-17-1A Paul et al., 1986 Surface GP CO17-1A LoBuglio etal., 1988 CEA ZCE-025 Patt et al., 1988 CEA AB2 Griffin et al., 1988acell surface AG HT-29-15 Cohn et al., 1987 secretory 250-30.6 Leydem etal., 1986 epithelium surface 44X14 Gallagher et al., 1986 glycoproteinNS A7 Takahashi et al., 1988 NS GA73.3 Munz et al., 1986 NS 791T/36Farrans et al., 1982 cell membrane & 28A32 Smith et al., 1987cytoplasmic Ag CEA & vindesine 28.19.8 Corvalen, 1987 gp72 X MMCO-791Byers et al., 1987 high M_(r) mucin DU-PAN-2 Lan et al., 1985 high M_(r)mucin ID₃ Gangopadhyay et al., 1985 CEA 180 Kd GP CEA 11-H5 Wagener etal., 1984 60 Kd GP 2C₈/2F₇ Bhattacharya et al., 1985 CA-19-9 (or CA-19-9(1116NS 19-9) Atkinson et al., 1982 GICA) Lewis a PR5C5 Imperial CancerResearch Technology Mab Listing Lewis a PR4D2 Imperial Cancer ResearchTechnology Mab Listing colonic mucus PR4D1 Imperial Cancer ResearchTechnology Mab Listing D: MELANOMA p97^(a) 4.1 Woodbury et al., 1980p97^(a) 8.2 M₁₇ Brown, et al., 1981a p97^(b) 96.5 Brown, et al., 1981ap97^(c) 118.1, 133.2, Brown, et al., 1981a (113.2) p97^(c) L₁, L₁₀,R₁₀(R₁₉) Brown et al., 1981b p97^(d) I₁₂ Brown et al., 1981b p97^(e) K₅Brown et al., 1981b p155 6.1 Loop et al., 1981 G_(D3) disialogan- R24Dippold et al., 1980 glioside p210, p60, p250 5.1 Loop et al., 1981 p280p440 225.28S Wilson et al., 1981 GP 94, 75, 70 & 465.12S Wilson et al.,1981 25 P240-P250, P450 9.2.27 Reisfeld et al., 1982 100, 77, 75 Kd F11Chee et al., 1982 94 Kd 376.96S Imai et al., 1982 4 GP chains 465.12SImai et al., 1982; Wilson et al., 1981 GP 74 15.75 Johnson &Reithmuller, 1982 GP 49 15.95 Johnson & Reithmuller, 1982 230 Kd Mel-14Carrel et al., 1982 92 Kd Mel-12 Carrel et al., 1982 70 Kd Me3-TB7Carrel et al., 1:387, 1982 HMW MAA similar 225.28SD Kantor et al., 1982to 9.2.27 AG HMW MAA similar 763.24TS Kantor et al., 1982 to 9.2.27 AGGP95 similar to 705F6 Stuhlmiller et al., 1982 376.96S 465.12S GP125436910 Saxton et al., 1982 CD41 M148 Imperial Cancer Research TechnologyMab listing E: high M_(r) mucin ID3 Gangopadhyay et al., 1985GASTROINTESTINAL pancreas, stomach gall bladder, high M_(r) mucinDU-PAN-2 Lan et al., 1985 pancreas, stomach pancreas NS OV-TL3 Poels etal., 1984 pancreas, stomach, ‘TAG-72’ high B72.3 Thor et al., 1986oesophagus M_(r) mucin stomach ‘CEA’ 180 Kd GP CEA 11-H5 Wagener et al.,1984 pancreas HMFG-2 > 400 Kd 3.14.A3 Burchell et al., 1983 GP G.I. NS CCOLI Lemkin et al., 1984 pancreas, stomach CA 19-9 (or CA-19-9 (1116NS19- Szymendera, 1986 GICA) 9) and CA50 pancreas CA125 GP OC125Szymendera, 1986 F: LUNG p185^(HER2) 4D5 3H4, 7C2, 6E9, Shepard et al.,1991 non-small cell 2C4, 7F3, 2H11, lung carcinoma 3E8, 5B8, 7D3, SB8high M_(r) mucin/ MO v2 Miotti et al., 1985 glycolipid ‘TAG-72’ highB72.3 Thor et al., 1986 M_(r) mucin high M_(r) mucin DU-PAN-2 Lan etal., 1985 ‘CEA’ 180 kD GP CEA 11-H5 Wagener et al., 1984 MalignantGliomas cytoplasmic MUC 8-22 Stavrou, 1990 antigen from 85HG-22 cellscell surface Ag MUC 2-63 Stavrou, 1990 from 85HG-63 cells cell surfaceAg MUC 2-39 Stavrou, 1990 from 86HG-39 cells cell surface Ag MUC 7-39Stavrou, 1990 from 86HG-39 cells G: MISCELLANEOUS p53 PAb 240 ImperialCancer Research Technology MaB PAb 246 Listing PAb 1801 small round cellneural cell ERIC.1 Imperial Cancer Research Technology MaB tumorsadhesion Listing molecule medulloblastoma M148 Imperial Cancer ResearchTechnology MaB neuroblastoma Listing rhabdomyosarcoma neuroblastomaFMH25 Imperial Cancer Research Technology MaB Listing renal cancer &p155 6.1 Loop et al., 1981 glioblastomas bladder & “Ca Antigen” CA1Ashall et al., 1982 laryngeal cancers 350-390 kD neuroblastoma GD2 3F8Cheung et al., 1986 Prostate gp48 48 kD GP 4F₇/7A₁₀ Bhattacharya et al.,1984 Prostate 60 kD GP 2C₈/2F₇ Bhattacharya et al., 1985 Thyroid ‘CEA’180 kD GP CEA 11-H5 Wagener et al., 1984 # pentaglycosyl ceramide orsialyated lacto-N-fucopentaose II; p97 Ags are believed to bechondroitin sulphate proteoglycan; antigens reactive with Mab 9.2.27 arebelieved to be sialylated glycoproteins associated with chondroitinsulphate proteoglycan; # unless specified, GY can include cancers of thecervix, endocervix, # endometrium, fallopian tube, ovary, vagina ormixed Mullerian tumor; unless specified GI can include cancers # of theliver, small intestine, spleen, pancreas, stomach and oesophagus.

[0117] TABLE II HUMAN TUMOR CELL LINES AND SOURCES ATTC HTB NUMBER CELLLINE TUMOR TYPE 1 J82 Transitional-cell carcinoma, bladder 2 RT4Transitional-cell papilloma, bladder 3 ScaBER Squamous carcinoma,bladder 4 T24 Transitional-cell carcinoma, bladder 5 TCCSUPTransitional-cell carcinoma, bladder, primary grade IV 9 5637 Carcinoma,bladder, primary 10 SK-N-MC Neuroblastoma, metastasis to supra-orbitalarea 11 SK-N-SH Neuroblastoma, metastasis to bone marrow 12 SW 1088Astrocytoma 13 SW 1783 Astrocytoma 14 U-87 MG Glioblastoma, astrocytoma,grade III 15 U-118 MG Glioblastoma 16 U-138 MG Glioblastoma 17 U-373 MGGlioblastoma, astrocytoma, grade III 18 Y79 Retinoblastoma 19 BT-20Carcinoma, breast 20 BT-474 Ductal carcinoma, breast 22 MCF7 Breastadenocarcinoma, pleural effusion 23 MDA-MB-134-VI Breast, ductalcarcinoma, pleural effusion 24 MDA-MD-157 Breast medulla, carcinoma,pleural effusion 25 MDA-MB-175-VII Breast, ductal carcinoma, pleuraleffusion 27 MDA-MB-361 Adenocarcinoma, breast, metastasis to brain 30SK-BR-3 Adenocarcinoma, breast, malignant pleural effusion 31 C-33 ACarcinoma, cervix 32 HT-3 Carcinoma, cervix, metastasis to lymph node 33ME-180 Epidermoid carcinoma, cervix, metastasis to omentum 34 MS751Epidermoid carcinoma, cervix, metastasis to lymph node 35 SiHa Squamouscarcinoma, cervix 36 JEG-3 Choriocarcinoma 37 Caco-2 Adenocarcinoma,colon 38 HT-29 Adenocarcinoma, colon, moderately well- differentiatedgrade II 39 SK-CO-1 Adenocarcinoma, colon, ascites 40 HuTu 80Adenocarcinoma, duodenum 41 A-253 Epidermoid carcinoma, submaxillarygland 43 FaDu Squamous cell carcinoma, pharynx 44 A-498 Carcinoma,kidney 45 A-704 Adenocarcinoma, kidney 46 Caki-1 Clear cell carcinoma,consistent with renal primary, metastasis to skin 47 Caki-2 Clear cellcarcinoma, consistent with renal primary 48 SK-NEP-1 Wilms' tumor,pleural effusion 49 SW 839 Adenocarcinoma, kidney 52 SK-HEP-1Adenocarcinoma, liver, ascites 53 A-427 Carcinoma, lung 54 Calu-1Epidermoid carcinoma grade III, lung, metastasis to pleura 55 Calu-3Adenocarcinoma, lung, pleural effusion 56 Calu-6 Anaplastic carcinoma,probably lung 57 SK-LU-1 Adenocarcinoma, lung consistent with poorlydifferentiated, grade III 58 SK-MES-1 Squamous carcinoma, lung, pleuraleffusion 59 SW 900 Squamous cell carcinoma, lung 60 EB1 Burkittlymphoma, upper maxilla 61 EB2 Burkitt lymphoma, ovary 62 P3HR-1 Burkittlymphoma, ascites 63 HT-144 Malignant melanoma, metastasis tosubcutaneous tissue 64 Malme-3M Malignant melanoma, metastasis to lung66 RPMI-7951 Malignant melanoma, metastasis to lymph node 67 SK-MEL-1Malignant melanoma, metastasis to lymphatic system 68 SK-MEL-2 Malignantmelanoma, metastasis to skin of thigh 69 SK-MEL-3 Malignant melanoma,metastasis to lymph node 70 SK-MEL-5 Malignant melanoma, metastasis toaxillary node 71 SK-MEL-24 Malignant melanoma, metastasis to node 72SK-MEL-28 Malignant melanoma 73 SK-MEL-31 Malignant melanoma 75 Caov-3Adenocarcinoma, ovary, consistent with primary 76 Caov-4 Adenocarcinoma,ovary, metastasis to subserosa of fallopian tube 77 SK-OV-3Adenocarcinoma, ovary, malignant ascites 78 SW 626 Adenocarcinoma, ovary79 Capan-1 Adenocarcinoma, pancreas, metastasis to liver 80 Capan-2Adenocarcinoma, pancrease 81 DU 145 Carcinoma, prostate, metastasis tobrain 82 A-204 Rhabdomyosarcoma 85 Saos-2 Osteogenic sarcoma, primary 86SK-ES-1 Anaplastic osteosarcoma versus Ewing sarcoma, bone 88 SK-LMS-1Leiomyosarcoma, vulva, primary 91 SW 684 Fibrosarcoma 92 SW 872Liposarcoma 93 SW 982 Axilla synovial sarcoma 94 SW 1353 Chondrosarcoma,humerus 96 U-2 OS Osteogenic sarcoma, bone primary 102 Malme-3 Skinfibroblast 103 KATO III Gastric carcinoma 104 Cate-1B Embryonalcarcinoma, testis, metastasis to lymph node 105 Tera-1 Embryonalcarcinoma, malignancy consistent with metastasis to lung 106 Tera-2Embryonal carcinoma, malignancy consistent with, metastasis to lung 107SW579 Thyroid carcinoma 111 AN3 CA Endometrial adenocarcinoma,metastatic 112 HEC-1-A Endometrial adenocarcinoma 113 HEC-1-BEndometrial adenocarcinoma 114 SK-UT-1 Uterine, mixed mesodermal tumor,consistent with leiomyosarcoma grade III 115 SK-UT-1B Uterine, mixedmesodermal tumor, consistent with leiomyosarcoma grade III 117 SW 954Squamous cell carcinoma, vulva 118 SW 962 Carcinoma, vulva, lymph nodemetastasis 119 NCI-H69 Small cell carcinoma, lung 120 NCI-H128 Smallcell carcinoma, lung 121 BT-483 Ductal carcinoma, breast 122 BT-549Ductal carcinoma, breast 123 DU4475 Metastatic cutaneous nodule, breastcarcinoma 124 HBL-100 Breast 125 Hs 578Bst Breast, normal 126 Hs 578TDuctal carcinoma, breast 127 MDA-MB-330 Carcinoma, breast 128 MDA-MB-415Adenocarcinoma, breast 129 MDA-MB-435S Ductal carcinoma, breast 130MDA-MB-436 Adenocarcinoma, breast 131 MDA-MB-453 Carcinoma, breast 132MDA-MB-468 Adenocarcinoma, breast 133 T-47D Ductal carcinoma, breast,pleural effusion 134 Hs 766T Carcinoma, pancreas, metastatic to lymphnode 135 Hs 746T Carcinoma, stomach, metastatic to left leg 137 Hs 695TAmelanotic melanoma, metastatic to lymph node 138 Hs 683 Glioma 140 Hs294T Melanoma, metastatic to lymph node 142 Hs 602 Lymphoma, cervical144 JAR Choriocarcinoma, placenta 146 Hs 445 Lymphoid, Hodgkin's disease147 Hs 700T Adenocarcinoma, metastatic to pelvis 148 H4 Neuroglioma,brain 151 Hs 696 Adenocarcinoma primary, unknown, metastatic tobone-sacrum 152 Hs 913T Fibrosarcoma, metastatic to lung 153 Hs 729Rhabdomyosarcoma, left leg 157 FHs 738Lu Lung, normal fetus 158 FHs173We Whole embryo, normal 160 FHs 738Bl Bladder, normal fetus 161 NIH:0VCAR-3 Ovary, adenocarcinoma 163 Hs 67 Thymus, normal 166 RD-ES Ewing'ssarcoma 168 ChaGo K-1 Bronchogenic carcinoma, subcutaneous metastasis,human 169 WERI-Rb-1 Retinoblastoma 171 NCI-H446 Small cell carcinoma,lung 172 NCI-H209 Small cell carcinoma, lung 173 NCI-H146 Small cellcarcinoma, lung 174 NCI-H441 Papillary adenocarcinoma, lung 175 NCI-H82Small cell carcinoma, lung 176 H9 T-cell lymphoma 177 NCI-H460 Largecell carcinoma, lung 178 NCI-H596 Adenosquamous carcinoma, lung 179NCI-H676B Adenocarcinoma, lung 180 NCI-H345 Small cell carcinoma, lung181 NCI-H820 Papillary adenocarcinoma, lung 182 NCI-H520 Squamous cellcarcinoma, lung 183 NCI-H661 Large cell carcinoma, lung 184 NCI-H510ASmall cell carcinoma, extra-pulmonary origin, metastatic 185 D283 MedMedulloblastoma 186 Daoy Medulloblastoma 187 D341 Med Medulloblastoma188 AML-193 Acute monocyte leukemia 189 MV4-11 Leukemia biphenotype

[0118] (a) Anti-Tumor Cell Antibodies

[0119] A straightforward means of recognizing a tumor antigen target isthrough the use of an antibody that has binding affinity for theparticular antigen. An extensive number of antibodies are known that aredirected against solid tumor antigens. Certain useful anti-tumorantibodies are listed above in Table I. However, as will be instantlyknown to those of skill in the art, certain of the antibodies listed inTable I will not have the appropriate biochemical properties, or may notbe of sufficient tumor specificity, to be of use therapeutically. Anexample is MUC8-22 that recognizes a cytoplasmic antigen. Antibodiessuch as these will generally be of use only in investigationalembodiments, such as in model systems or screening assays.

[0120] Generally speaking, antibodies for use in these aspects of thepresent invention will preferably recognize antigens that are accessibleon the cell-surface and that are preferentially, or specifically,expressed by tumor cells. Such antibodies will also preferably exhibitproperties of high affinity, such as exhibiting a K_(d) of <200 nM, andpreferably, of <100 nM, and will not show significant reactivity withlife-sustaining normal tissues, such as one or more tissues selectedfrom heart, kidney, brain, liver, bone marrow, colon, breast, prostate,thyroid, gall bladder, lung, adrenals, muscle, nerve fibers, pancreas,skin, or other life-sustaining organ or tissue in the human body. The“life-sustaining” tissues that are the most important for the purposesof the present invention, from the standpoint of low reactivity, includeheart, kidney, central and peripheral nervous system tissues and liver.The term “significant reactivity”, as used herein, refers to an antibodyor antibody fragment, that, when applied to the particular tissue underconditions suitable for immunohistochemistry, will elicit either nostaining or negligible staining with only a few positive cells scatteredamong a field of mostly negative cells.

[0121] Particularly promising antibodies from Table I contemplated foruse in the present invention are those having high selectivity for thesolid tumor. For example, antibodies binding to TAG 72 and the HER-2proto-oncogene protein, which are selectively found on the surfaces ofmany breast, lung and colorectal cancers (Thor et al., 1986; Colcher etal., 1987; Shepard et al., 1991); MOv18 and OV-TL3 and antibodies thatbind to the milk mucin core protein and human milk fat globule (Miottiet al., 1985; Burchell et al., 1983); and the antibody 9.2.27 that bindsto the high M_(r) melanoma antigens (Reisfeld et al., 1982). Furtheruseful antibodies are those against the folate-binding protein, which isknown to be homogeneously expressed in almost all ovarian carcinomas;those against the erb family of oncogenes that are over-expressed insquamous cell carcinomas and the majority of gliomas; and otherantibodies known to be the subject of ongoing pre-clinical and clinicalevaluation.

[0122] The antibodies B3, KSI/4, CC49, 260F9, XMMCO-791, D612 and SM3are believed to be particularly suitable for use in clinicalembodiments, following the standard pre-clinical testing routinelypracticed in the art. B3 (U.S. Pat. No. 5,242,813; Brinkmann et al.,1991) has ATCC Accession No. HB 10573; KS1/4 can be made as described inU.S. Pat. No. 4,975,369; and D612 (U.S. Pat. No. 5,183,756) has ATCCAccession No. HB 9796.

[0123] Another means of defining a tumor-associated target is in termsof the characteristics of the tumor cell, rather than describing thebiochemical properties of an antigen expressed by the cell. Accordingly,the inventors contemplate that any antibody that preferentially binds toa tumor cell listed in Table II may be used as the targeting componentof a bispecific ligand. The preferential tumor cell binding is againbased upon the antibody exhibiting high affinity for the tumor cell andnot having significant reactivity with life-sustaining normal cells ortissues, as defined above.

[0124] The invention therefore provides several means for generating anantibody for use in the targeted coagulation methods described herein.To generate a tumor cell-specific antibody, one would immunize an animalwith a composition comprising a tumor cell antigen and, as describedmore fully in below, select a resultant antibody with appropriatespecificity. The immunizing composition may contain a purified, orpartially purified, preparation of any of the antigens in Table I; acomposition, such as a membrane preparation, enriched for any of theantigens in Table I; any of the cells listed in Table II; or a mixtureor population of cells that include any of the cell types listed inTable II.

[0125] Of course, regardless of the source of the antibody, in thepractice of the invention in human treatment, one will prefer to ensurein advance that the clinically-targeted tumor expresses the antigenultimately selected. This is achieved by means of a fairlystraightforward assay, involving antigenically testing a tumor tissuesample, for example, a surgical biopsy, or perhaps testing forcirculating shed antigen. This can readily be carried out in animmunological screening assay such as an ELISA (enzyme-linkedimmunosorbent assay), wherein the binding affinity of antibodies from a“bank” of hybridomas are tested for reactivity against the tumor.Antibodies demonstrating appropriate tumor selectivity and affinity arethen selected for the preparation of bispecific antibodies of thepresent invention.

[0126] Due to the well-known phenomenon of cross-reactivity, it iscontemplated that useful antibodies may result from immunizationprotocols in which the antigens originally employed were derived from ananimal, such as a mouse or a primate, in addition to those in which theoriginal antigens were obtained from a human cell. Where antigens ofhuman origin are used, they may be obtained from a human tumor cellline, or may be prepared by obtaining a biological sample from aparticular patient in question. Indeed, methods for the development ofantibodies that are “custom-tailored” to the patient's tumor are known(Stevenson et al., 1990) and are contemplated for use in connection withthis invention.

[0127] (b) Further Tumor Cell Targets and Binding Ligands

[0128] In addition to the use of antibodies, other ligands could beemployed to direct a coagulating agent to a tumor site by binding to atumor cell antigen. For tumor antigens that are over-expressed receptors(oestrogen receptor, EGF receptor), or mutant receptors, thecorresponding ligands could be used as targeting agents.

[0129] In an analogous manner to endothelial cell receptor ligands,there may be components that are specifically, or preferentially, boundto tumor cells. For example, if a tumor antigen is an over-expressedreceptor, the tumor cell may be coated with a specific ligand in vivo.It seems that the ligand could then be targeted either with an antibodyagainst the ligand, or with a form of the receptor itself. Specificexamples of these type of targeting agents are antibodies against TIE-1or TIE-2 ligands, antibodies against platelet factor 4, and leukocyteadhesion binding protein.

[0130] 2. Other Disease Targets

[0131] In further embodiments, the first binding region may be acomponent that binds to a target molecule that is specifically orpreferentially expressed in a disease site other than a tumor site.

[0132] Exemplary target molecules associated with other diseased cellsinclude, for example, leukocyte adhesion molecules, that are associatedwith psoriasis; FGF, that is associated with proliferative diabeticretinopathy; platelet factor 4, that is associated with the activatedendothelium of various diseases; and VEGF, that is associated withvascular proliferative disease. It is believed that an animal or patienthaving any one of the above diseases would benefit from the specificinduction of coagulation in the disease site.

[0133] Diseases that are known to have a common angio-dependentpathology, as described in Klagsburn & Folkman (1990), may also betreated with bispecific ligand as described herein. In particular, avascular endothelial cell-targeted ligand or a stroma-targeted ligandwill be used to achieve coagulation in the disease site. The treatmentof BPH, diabetic retinopathy, vascular restenosis, vascular adhesions,AVM, meningioma, hemangioma, neovascular glaucoma, rheumatoid arthritisand psoriasis are particularly contemplated at the present time.

[0134] 3. Disease-Associated Vasculature Cell Targets

[0135] The cells of the vasculature are intended as targets for use inthe present invention. In these cases, one binding region of thebispecific ligand will be capable of binding to an accessible markerpreferentially expressed by disease-associated vasculature endothelialcells. The exploitation of the vascular markers is made possible due tothe proximity of the vascular endothelial cells to the disease area andto the products of the local aberrant physiological processes. Forexample, tumor vascular endothelial cells are exposed to tumor cells andtumor-derived products that change the phenotypic profile of theendothelial cells.

[0136] Tumor cells are known to elaborate tumor-derived products, suchas lymphokines, monokines, colony-stimulating factors, growth factorsand angiogenic factors, that act on the nearby vascular endothelialcells (Kandel et al., 1991; Folkman, 1985a,b) and cytokines (Burrows etal., 1991; Ruco et al., 1990; Borden et al., 1990). The tumor productsbind to the endothelial cells and serve to selectively induce expressionof certain molecules. It is these induced molecules that may be targetedusing the tumor endothelium-specific coagulant delivery provided bycertain aspects of the present invention. Vascular endothelial cells intumors proliferate at a rate 30-fold greater than those in miscellaneousnormal tissues (Denekamp et al., 1982), suggesting thatproliferation-linked determinants could also serve as markers for tumorvascular endothelial cells.

[0137] In certain embodiments of the invention, the targeting componentof the bispecific ligands will be a component that has a relatively highdegree of specificity for tumor vasculature. These targeting componentsmay be defined as components that bind to molecules expressed on tumorendothelium, but that have little or no expression at the surface ofnormal endothelial cells. Such specificity may be assessed by thestandard procedures of immunostaining of tissue sections, which areroutine to those of skill in the art.

[0138] However, as stated above, an advantage of the present inventionis that the requirement for selectivity is not as stringent aspreviously needed in the prior art methods, especially those employingimmunotoxins, because any side effects associated with the mis-targetingof the coagulating agent will be minimal in comparison to thoseresulting from the mis-targeting of a toxin.

[0139] Therefore, it is generally proposed that the molecules to betargeted using the bispecific ligands or antibodies of this inventionwill be those that are expressed on tumor vasculature at a higher levelthan on normal endothelial cells.

[0140] (a) Vascular Endothelial Cell Markers in Disease

[0141] Molecules that are known to be preferentially expressed at thesurface of vascular endothelial cells in a disease site or environmentare herein termed “natural disease-associated vascular endothelial cellmarkers”. This term is used for simplicity to refer to the endothelialcell components that are expressed in diseases connected with increasedor inappropriate angiogenesis or endothelial cell proliferation. Oneparticular example are the tumor endothelial cell components that areexpressed in situ in response to tumor-derived factors. These componentsare also termed “naturally-induced tumor endothelial cell markers”.

[0142] Both VEGF/VPF (vascular endothelial growth factor/vascularpermeability factor) and components of the FGF (fibroblast growthfactor) family are concentrated in or on tumor vasculature. Thecorresponding receptors therefore provide a potential target for attackon tumor vasculature. For example, VEGF receptors are known to beupregulated on tumor endothelial cells, as opposed to endothelial cellsin normal tissues, both in rodents and man (Thieme et al., 1995).Possibly, this is a consequence of hypoxia—a characteristic of the tumormicroenvironment (Leith et al., 1992). FGF receptors are alsoupregulated three-fold on endothelial cells exposed to hypoxia, and soare believed to be upregulated in tumors (Bicknell and Harris et al.,1992).

[0143] The TGFβ (transforming growth factor β) receptor (endoglin) onendothelial cells is upregulated on dividing cells, providing anothertarget. One of the present inventors found that endoglin is upregulatedon activated and dividing HUVEC in culture, and is strongly expressed inhuman tissues on endothelial cells at sites of neovascularization,including a broad range of solid tumors and fetal placenta. In contrast,endothelial cells in the majority of miscellaneous non-malignant adulttissues, including preneoplastic lesions, contain little or no endoglin.Importantly, endoglin expression is believed to correlate withneoplastic progression in the breast, as shown by benign fibroadenomasand early carcinomas binding low levels of TEC-4 and TEC-11 antibodies,and late stage intraductal carcinomas and invasive carcinomas bindinghigh levels of these antibodies.

[0144] Other natural disease-associated vascular endothelial cell,markers include a TIE, VCAM-1, P-selectin, E-selectin, α_(v)β₃ integrin,pleiotropin and endosialin, each of which may be targeted using theinvention.

[0145] (b) Cytokine-Inducible Vascular Endothelial Markers

[0146] Due to the nature of disease processes, which often result inlocalized dysfunction within the body, methods are available tomanipulate the disease site whilst leaving other tissues relativelyunaffected. This is particularly true in malignant and benign tumors,which exist as distinct entities within the body of an animal. Forexample, the tumor environment may be manipulated to create additionalmarkers that are specific for tumor vascular endothelial cells. Thesemethods generally mimic those that occur naturally in solid tumors, andalso involve the local production of signalling agents, such as growthfactors or cytokines, that induce the specific expression of certainmolecules at the surface of the nearby vascular endothelial cells.

[0147] The group of molecules that may be artificially induced to beexpressed at the surface of vascular endothelial cells in a disease ortumor environment are herein termed “inducible endothelial cellmarkers”, or specifically, inducible tumor endothelial cell markers.This term is used to refer to those markers that are artificiallyinduced, i.e., induced as a result of manipulation by the hand of man,rather than those that are induced as part of the disease or tumordevelopment process in an animal. The term “inducible marker”, asdefined above, is chosen for simple reference in the context of thepresent application, notwithstanding the fact that “natural markers” arealso induced, e.g., by tumor-derived agents.

[0148] Thus, although not required to practice the invention, techniquesfor the selective elicitation of vascular endothelial antigen targets onthe surface of disease-associated vasculature are available that may, ifdesired, be used in conjunction with the invention. These techniquesinvolve manipulating the antigenic expression, or cell surfacepresentation, such that a target antigen is expressed or renderedavailable on the surface of disease-associated vasculature and notexpressed or otherwise rendered accessible or available for binding, orat least to a lesser extent, on the surface of normal endothelium.

[0149] Tumor endothelial markers can be induced by tumor-derivedcytokines (Burrows et al., 1991; Ruco et al., 1990) and by angiogenicfactors (Mignatti et al., 1991). Examples of cell surface markers thatmay be specifically induced in the tumor endothelium and then targetedusing a bispecific coagulating ligand, as provided by the invention,include those listed in Table III (Bevilacqua et al., 1987; Dustin etal., 1986; Osborn et al., 1989; Collins et al., 1984).

[0150] The mechanisms for the induction of the proposed markers; theinducing, or “intermediate cytokine”, such as IL-1 and IFN-γ; and theleukocyte cell type and associated cytokine-activating molecule, whosetargeting will result in the release of the cytokine, are also set forthin Table III. In the induction of a specific marker, a bispecific“cytokine-inducing” or “antigen-inducing” antibody is generallyrequired. This antibody will selectively induce the release of theappropriate cytokine in the locale of the tumor, thus selectivelyinducing the expression of the desired target antigen by the vascularendothelial cells. The bispecific antibody cross-links cells of thetumor mass and cytokine-producing leukocytes, thereby activating theleukocytes to release the cytokine.

[0151] The preparation and use of bispecific antibodies such as these ispredicated in part on the fact that cross-linking antibodies recognizingCD3, CD14, CD16 and CD28 have previously been shown to elicit cytokineproduction selectively upon cross-linking with the second antigen (Qianet al., 1991). In the context of the present invention, since onlysuccessfully tumor cell-crosslinked leukocytes will be activated torelease the cytokine, cytokine release will be restricted to the localeof the tumor. Thus, expression of the desired marker, such asE-selectin, will be similarly limited to the endothelium of the tumorvasculature. TABLE III POSSIBLE INDUCIBLE VASCULAR TARGETS LEUKOCYTEMOLECULES WHICH, WHEN CROSSLINKED BY INDUCIBLE SUBTYPES/ALIASESLEUKOCYTES WHICH MONOCLONAL ANTIBODIES ENDOTHELIAL (MOLECULAR INDUCINGPRODUCE THOSE ACTIVATE THE CELLS TO CELL MOLECULES ACRONYM FAMILY)CYTOKINES CYTOKINES PRODUCE CYTOKINES Endothelial- ELAM-1 — IL-1, TNF-monocytes CD14 Leukocyte E- (Selectin) α, (TNF-β) macrophages CD14Adhesion select- (Bacterial mast cells FcR for IgE Molecule-1 inEndotoxin) Vascular Cell VCAM-1 Inducible Cell (Bacterial monocytes CD14Adhesion Adhesion Endotoxin) macrophages CD14 Molecule-1 Molecule-110IL-1, TNF-α mast cells FcR for IgE (INCAM-110) TNF-β, IL-4 helper Tcells CD2, CD3, CD28 (Immunoglobulin TNF NK cells FcR for IgG (CD16)Family) Intercellular ICAM-1 — IL-1, TNFα monocytes CD14 Adhesion(Immunoglobulin (Bacterial macrophages CD15 Molecule-1 Family)Endotoxin) mast cells FcR for IgE TNF-β, T helper cells CD2, CD3, CD28IFNγ NK cells FcR for IgG (CD16) The Agent for LAM-1 MEL-14 Agent Il-1,TNFα monocytes CD14 Leukocyte Agent (Mouse) (Bacterial macrophages CD14Adhesion Endotoxin) mast cells FcR for IgE Molecule-1 Major MHC HLA-DRIFN-γ helper T cells CD2, CD3, CD28 Histocompatability Class HLA-DP -Human Complex II HLA-DQ Class II I-A - Mouse NK cells FcR for IgG (CD16)Antigen I-E

[0152] It is important to note that, from the possible inducible markerslisted in Table III, E-selectin and MHC Class II antigens, such asHLA-DR, HLA-DP and HLA-DQ (Collins et al., 1984), are by far the mostpreferred targets for use in connection with clinical embodiments. Theother adhesion molecules of Table III appear to be expressed to varyingdegrees in normal tissues, generally in lymphoid organs and onendothelium, making their targeting perhaps appropriate only in animalmodels or in cases where their expression on normal tissues can beinhibited without significant side-effects. The targeting of E-selectinor an MHC Class II antigen is preferred as the expression of theseantigens will likely be the most direct to promote selectively intumor-associated endothelium.

[0153] E-Selectin

[0154] The targeting of an antigen that is not expressed on the surfacesof normal endothelium is the most straightforward form of the inductionmethods. E-selectin is an adhesion molecule that is not expressed innormal endothelial vasculature or other human cell types (Cotran et al.,1986), but can be induced on the surface of endothelial cells throughthe action of cytokines such as IL-1, TNF, lymphotoxin and bacterialendotoxin (Bevilacqua et al., 1987). It is not induced by IFN-γ (Wu etal., 1990). The expression of E-selectin may thus be selectively inducedin tumor endothelium through the selective delivery of such a cytokine,or via the use of a composition that causes the selective release ofsuch cytokines in the tumor environment.

[0155] Bispecific antibodies are one example of a composition capable ofcausing the selective release of one or more of the foregoing or otherappropriate cytokines in the tumor site, but not elsewhere in the body.Such bispecific antibodies are herein termed “antigen-inducingantibodies” and are, of course, distinct from any bispecific antibodiesof the invention that have targeting and coagulating components.Antigen-inducing antibodies are designed to cross-link cytokine effectorcells, such as cells of monocyte/macrophage lineage, T cells and/or NKcells or mast cells, with tumor cells of the targeted solid tumor mass.This cross-linking would then effect a release of cytokine that islocalized to the site of cross-linking, i.e., the tumor.

[0156] Effective antigen-inducing antibodies recognize a selected tumorcell surface antigen on the one hand (e.g., those in Table I) and, onthe other hand, recognize a selected “cytokine activating” antigen onthe surface of a selected leukocyte cell type. The term “cytokineactivating” antigen is used to refer to any one of the various knownmolecules on the surfaces of leukocytes that, when bound by an effectormolecule, such as an antibody or a fragment thereof or anaturally-occurring agent or synthetic analog thereof, be it a solublefactor or membrane-bound counter-receptor on another cell, promotes therelease of a cytokine by the leukocyte cell. Examples of cytokineactivating molecules include CD14 (the LPS receptor) and FcR for IgE,which will activate the release of IL-1 and TNFα; and CD16, CD2 or CD3or CD28, which will activate the release of IFNγ and TNFβ, respectively.

[0157] Once introduced into the bloodstream of an animal bearing atumor, such an antigen-inducing bispecific antibody will bind to tumorcells within the tumor, cross-link those tumor cells with effectorcells, e.g., monocytes/macrophages, that have infiltrated the tumor, andthereafter effect the selective release of cytokine within the tumor.Importantly, however, without cross-linking of the tumor and leukocyte,the antigen-inducing antibody will not effect the release of cytokine.Thus, no cytokine release will occur in parts of the body removed fromthe tumor and, hence, expression of cytokine-induced molecules, e.g.,E-selectin, will occur only within the tumor endothelium.

[0158] A number of useful “cytokine activating” antigens are known,which, when cross-linked with an appropriate bispecific antibody, willresult in the release of cytokines by the cross-linked leukocyte. Thegenerally preferred target for this purpose is CD14, which is found onthe surface of monocytes and macrophages. When CD14 is cross linked itstimulates monocytes/macrophages to release IL-1 (Schutt et al., 1988;Chen et al., 1990), and possibly other cytokines, which, in turnstimulate the appearance of E-selectin on nearby vasculature. Otherpossible targets for cross-linking in connection with E-selectininduction and targeting include FcR for IgE, found on Mast cells; FcRfor IgG (CD16), found on NK cells; as well as CD2, CD3 or CD28, found onthe surfaces of T cells. Of these, CD14 targeting is generally preferreddue to the relative prevalence of monocyte/macrophage infiltration ofsolid tumors as opposed to the other leukocyte cell types.

[0159] In an exemplary induction embodiment, an animal bearing a solidtumor is injected with bispecific (Fab′-Fab′) anti-CD14/anti-tumorantibody (such as anti-CEA, 9.2.27 antibody against high Mr melanomaantigens OV-TL3 or MOv 18 antibodies against ovarian associatedantigens). The antibody localizes in the tumor, by virtue of its tumorbinding activity, and then activates monocytes and macrophages in thetumor by crosslinking their CD14 antigens (Schutt et. al., 1988; Chenet. al., 1990). The activated monocytes/macrophages have tumoricidalactivity (Palleroni et. al., 1991) and release IL-1 and TNF whichrapidly induce E-selectin antigens on the tumor vascular endothelialcells (Bevilacqua et. al., 1987; Pober et. al., 1991).

[0160] MHC Class II Antigens

[0161] The second preferred group of inducible markers contemplated foruse with the present invention are the MHC Class II antigens (Collins etal., 1984), including HLA-DR, HLA-DP and HLA-DQ. Class II antigens areexpressed on vascular endothelial cells in most normal tissues inseveral species, including man. Studies in vitro (Collins et al., 1984;Daar et al., 1984; O'Connell et al., 1990) and in vivo (Groenewegen etal., 1985) have shown that the expression of Class II antigens byvascular endothelial cells requires the continuous presence of IFN-γwhich is elaborated by T_(H1) cells and, to a lesser extent, by NK cellsand CD8⁺ T cells.

[0162] MHC Class II antigens are not unique to vascular endothelialcells, and are also expressed constitutively on B cells, activated Tcells, cells of monocyte/macrophage linage and on certain epithelialcells, both in mice (Hammerling, 1976) and in man (Daar et al., 1984).Due to the expression of MHC Class II antigens on “normal” endothelium,their targeting is not quite so straightforward as E-selectin. However,the induction and targeting of MHC Class II antigens is made possible byusing in conjunction with an immunosuppressant, such as Cyclosporin A(CsA), that has the ability to effectively inhibit the expression ofClass II molecules in normal tissues (Groenewegen et al., 1985). The CsAacts by preventing the activation of T cells and NK cells (Groenewegenet al., 1985; DeFranco, 1991), thereby reducing the basal levels ofIFN-γ below those needed to maintain Class II expression on endothelium.

[0163] There are various other cyclosporins related to CsA, includingcyclosporins A, B, C, D, G, and the like, that also haveimmunosuppressive action and are likely to demonstrate an ability tosuppress Class II expression. Other agents that might be similarlyuseful include FK506 and rapamycin.

[0164] Thus, the practice of the MHC Class II induction and targetingembodiment requires a pretreatment of the tumor-bearing animal with adose of CsA or other Class II immunosuppressive agent that is effectiveto suppress Class II expression. In the case of CsA, this will typicallybe on the order of about 10 to about 30 mg/kg body weight. Oncesuppressed in normal tissues, Class II antigens can then be selectivelyinduced in the tumor endothelium, again through the use of a bispecificantibody.

[0165] In this case, the antigen-inducing bispecific antibody will havespecificity for a tumor cell marker and for an activating antigen foundon the surface of an effector cell that is capable of inducing IFN-γproduction. Such effector cells will generally be helper T cells (T_(H))or Natural Killer (NK) cells. In these embodiments, it is necessary thatT cells, or NK cells if CD16 is used, be present in the tumor to producethe cytokine intermediate in that Class II antigen expression isachieved using IFN-γ, but is not achieved with the other cytokines.Thus, for the practice of this aspect of the invention, one will desireto select CD2, CD3, CD28, or most preferably CD28, as the cytokineactivating antigen for targeting by the antigen-inducing bispecificantibody.

[0166] The T cells that should be activated in the tumor are thoseadjacent to the vasculature since this is the region most accessible tocells and is also where the bispecific antibody will be mostconcentrated. The activated T cells should then secrete IFN-γ whichinduces Class II antigens on the adjacent tumor vasculature.

[0167] The use of a bispecific (Fab′-Fab′) antibody having one armdirected against a tumor antigen and the other arm directed against CD28is currently preferred. This antibody will crosslink CD28 antigens on Tcells in the tumor which, when combined with a second signal (provided,for example, by IL-1 which is commonly secreted by tumor cells (Burrowset al., 1991; Ruco et al., 1990), has been shown to activate T cellsthrough a CA²⁺-independent non-CsA-inhibitable pathway (Hess et al.,1991; June et al., 1987; Bjorndahl et al., 1989).

[0168] The preparation of antibodies against various cytokine activatingmolecules is also well known in the art. For example, the preparationand use of anti-CD14 and anti-CD28 monoclonal antibodies having theability to induce cytokine production by leukocytes has now beendescribed by several laboratories (reviewed in Schutt et al., 1988; Chenet al., 1990, and June et al., 1990, respectively). Moreover, thepreparation of monoclonal antibodies that will stimulate leukocyterelease of cytokines through other mechanisms and other activatingantigens is also known (Clark et al., 1986; Geppert et al., 1990).

[0169] In still further embodiments, the inventors contemplate analternative approach for suppressing the expression of Class IImolecules, and selectively eliciting Class II molecule expression in thelocale of the tumor. This approach, which avoids the use of both CsA anda bispecific activating antibody, takes advantage of the fact that theexpression of Class II molecules can be effectively inhibited bysuppressing IFN-γ production by T cells, e.g., through use of ananti-CD4 antibody (Street et al., 1989). Using this embodiment, IFN-γproduction is inhibited by administering anti-CD4, resulting in thegeneral suppression of Class II expression. Class II is then inducedonly in the tumor site, e.g., using tumor-specific T cells which areonly activatable within the tumor.

[0170] In this mode of treatment, one will generally pretreat an animalor human patient with a dose of anti-CD4 that is effective to suppressIFN-γ production and thereby suppress the expression of Class IImolecules. Effective doses are contemplated to be, for example, on theorder of about 4 to about 10 mg/kg body weight. After Class IIexpression is suppressed, one will then prepare and introduce into thebloodstream an IFN-γ-producing T cell clone (e.g., T_(h)1 or cytotoxic Tlymphocyte, CTL) specific for an antigen expressed on the surface of thetumor cells. These T cells localizes to the tumor mass, due to theirantigen recognition capability and, upon such recognition, then releaseIFN-γ. In this manner, cytokine release is again restricted to thetumor, thus limiting the expression of Class II molecules to the tumorvasculature.

[0171] The IFN-γ-producing T cell clone may be obtained from theperipheral blood (Mazzocchi et al., 1990), however, a preferred sourceis from within the tumor mass (Fox et al., 1990). The currentlypreferred means of preparing such a T cell clone is to remove a portionof the tumor mass from a patient; isolate cells, using collagenasedigestion, where necessary; enrich for tumor infiltrating leukocytesusing density gradient centrifugation, followed by depletion of otherleukocyte subsets by, e.g., treatment with specific antibodies andcomplement; and then expand the tumor infiltrating leukocytes in vitroto provide the IFN-γ producing clone. This clone will necessarily beimmunologically compatible with the patient, and therefore should bewell tolerated by the patient.

[0172] It is proposed that particular benefits will be achieved byfurther selecting a high IFN-γ producing T cell clone from the expandedleukocytes by determining the cytokine secretion pattern of eachindividual clone every 14 days. To this end, rested clones will bemitogenically or antigenically-stimulated for about 24 hours and theirculture supernatants assayed, e.g., using a specific sandwich ELISAtechnique (Cherwinski et al., 1989), for the presence of IL-2, IFN-γ,IL-4, IL-5 and IL-10. Those clones secreting high levels of IL-2 andIFN-γ, the characteristic cytokine secretion pattern of T_(H1) clones,will be selected. Tumor specificity will be confirmed usingproliferation assays.

[0173] Furthermore, one will prefer to employ as the anti-CD4 antibodyan anti-CD4 Fab, because it will be eliminated from the body within 24hours after injection and so will not cause suppression of thetumor-recognizing T-cell clones that are subsequently administered. Thepreparation of T cell clones having tumor specificity is generally knownin the art, as exemplified by the production and characterization of Tcell clones from lymphocytes infiltrating solid melanoma tumors (Maedaet al., 1991).

[0174] In using either of the MHC Class II suppression-inductionmethods, additional benefits will likely result from the fact thatanti-Class II antibodies injected intravenously do not appear to reachthe epithelial cells or the monocytes/macrophages in normal organs otherthan the liver and spleen. Presumably this is because the vascularendothelium in most normal organs is tight, not fenestrated as it is inthe liver and spleen, and so the antibodies must diffuse across basementmembranes to reach the Class II-positive cells. Also, any B cellelimination that may result, e.g., following cross-linking, is unlikelyto pose a significant problem as these cells are replenished from ClassII negative progenitors (Lowe et al., 1986). Even B cell killing, asoccurs in B lymphoma patients, causes no obvious harm (Vitetta et al.,1991).

[0175] In summary, although the tumor coagulating compositions andantibodies of the present invention are elegantly simple, and do notrequire the induction of antigens for their operability, the combineduse of an antigen-inducing bispecific antibody with this invention isalso contemplated. Such antibodies would generally be administered priorto the bispecific coagulating ligands of this invention.

[0176] Generally speaking, the more “immunogenic” tumors would be moresuitable for the MHC Class II approach involving, e.g., thecross-linking of T cells in the tumor through an anti-CD28/anti-tumorbispecific antibody, because these tumors are more likely to beinfiltrated by T cells, a prerequisite for this method to be effective.Examples of immunogenic solid tumors include renal carcinomas,melanomas, a minority of breast and colon cancers, as well as possiblypancreatic, gastric, liver, lung and glial tumor cancers. These tumorsare referred to as “immunogenic” because there is evidence that theyelicit immune responses in the host and they have been found to beamenable to cellular immunotherapy (Yamaue et al., 1990). In the case ofmelanomas and large bowel cancers, the most preferred antibodies for usein these instances would be B72.3 (anti-TAG-72) and PRSC5/PR4C2(anti-Lewis a) or 9.2.27 (anti-high Mr melanoma antigen).

[0177] For the majority of solid tumors of all origins, an anti-CD14approach that employs a macrophage/monocyte intermediate would be moresuitable. This is because most tumors are rich in macrophages. Examplesof macrophage-rich tumors include most breast, colon and lungcarcinomas. Examples of preferred anti-tumor antibodies for use in theseinstances would be anti-HER-2, B72.3, SM-3, HMFG-2, and SWA11 (Smith etal., 1989).

[0178] (c) Coagulant-Inducible Markers

[0179] Coagulants, such as thrombin, Factor IX/IXa, Factor X/Xa, plasminand metalloproteinases, such as interstitial collagenases, stromelysinsand gelatinases, also act to induce certain markers. In particular,E-selectin, P-selectin, PDGF and ICAM-1 are induced by thrombin (Sugamaet. al., 1992; Shankar et. al., 1994).

[0180] Therefore, for this induction, an anti-coagulant/anti-tumorbispecific antibody will be utilized. The antibody will localize in thetumor via its tumor binding activity. The bispecific will thenconcentrate the coagulant, e.g., thrombin, in the tumor, resulting ininduction of E-selectin and P-selectin on the tumor vascular endothelialcells (Sugama et. al., 1991; Shankar et. al., 1994).

[0181] Alternatively, targeting of truncated tissue factor to tumorcells or endothelium will induce thrombin deposition within the tumor.As the thrombin is deposited, E-selectin and P-selectin will be inducedon the tumor vascular endothelial cells.

[0182] (d) Antibodies to Vascular Endothelial Cell Markers

[0183] A straightforward means of recognizing a disease-associatedvasculature target, whether induced in the natural environment or byartificial means, is through the use of an antibody that has bindingaffinity for the particular cell surface receptor, molecule or antigen.These include antibodies directed against all cell surface componentsthat are known to be present on, e.g., tumor vascular endothelial cells,those that are induced or over-expressed in response to tumor-derivedfactors, and those, that are induced following manipulation by the handof man. Table IV and Table V summarize useful antibodies and theirproperties. TABLE IV SUMMARY OF VASCULATURE STAINING PATTERNS OF CERTAINANTIBODIES TO HUMAN TUMOR VASCULATURE % Tumor % tumor normal typesvessels vessel Antibody Antigen Reference stained stained reactivityanti-vWF VIII R Ag 100 100 strong on all FB5 endosialin Rettig & 3010-20 lymphoid organs old TP3 80 kDa osteosarcoma Bruland 50 10-30strong on small related antigen BV protein BC-1 fibronectin isoformZardi 60 10-30 none TV-1 fibronectin Epstein 100 100 strong on all LM609 α_(v)β_(e) vitronectin Cheneoh 85 70-80 medium on all receptor TEC11 endoglin Thorpe; _(—) 100 100 weak on most TEC 110 VEGF Thorpe; _(—)100 100 weak on most

[0184] TABLE V COMPARISON OF ANTI-EC mAbs ON HUMAN TUMORS TUMOR TYPE nTEC 110 TEC 11 FB-5 TP-3 BC-1 TV-1 LM 609 DIGESTIVE Gastrointestinal 9++ ++ +−− ++ + ++ ++ Parotid 3 ++ ++ − ++ (SMALL) − ND ND REPRODUCTIVEBreast 1 + ++ − ND ++ ++ − Ovary 4 ++ ++ − ++ (SMALL) ++ ++ + Uterus 2++ ++ − ++ ++ + RESPIRATORY Lung 3 ++ ++ + ND ++ ++ + LYMPHOID Hodgkins2 ++ ++ − + − +−++ +

[0185] Two further antibodies that may be used in this invention arethose described by Rettig et al. (1992) and Wang et al. (1993) that aredirected against unrelated antigens of unknown function expressed in thevasculature of human tumors, but not in most normal tissues.

[0186] The antibody described by Kim et. al. (1993) may also be used inthis invention, particularly as this antibody inhibited angiogenesis andsuppressed tumor growth in vivo.

[0187] Antibodies that have not previously been shown to be specific forhuman tumors may also be used. For example, Venkateswaran et al. (1992)described the production of anti-FGF MAbs. Xu et. al. (1992) developedand characterized a panel of 16 isoform and domain-specific polyclonaland monoclonal antibodies against FGF receptor (flg) isoforms. Massogliaet al. (1987) also reported MAbs against the FGF receptor.

[0188] (e) Generation of Antibodies to Disease Vasculature

[0189] In addition to utilizing a known antibody, such as thosedescribed above and others known and published in the scientificliterature, one may also generate a novel antibody using standardimmunization procedures, as described in more detail hereinbelow. Togenerate an antibody against a known disease-associated vascular markerantigen, one would immunize an animal with an immunogenic compositioncomprising the antigen. This may be a membrane preparation thatincludes, or is enriched for, the antigen; a relatively purified form ofthe antigen, as isolated from cells or membranes; a highly purified formof the antigen, as obtained by a variety of purification steps using,e.g., a native antigen extract or a recombinant form of the antigenobtained from a recombinant host cell.

[0190] The present invention also provides yet further methods forgenerating an antibody against an antigen present on disease-associatedvasculature endothelial cells, which methods are suitable for use evenwhere the biochemical identity of the antigen remains unknown. Thesemethods are exemplified through the generation of an antibody againsttumor vasculature endothelial cells. A first means of achieving antibodygeneration in this manner uses a preparation of vascular endothelialcells obtained from the tumor site of an animal or human patient. Onesimply immunizes an experimental animal with a preparation of such cellsand collects the antibodies so produced. The most useful form of thismethod is that where specific antibodies are subsequently selected, asmay be achieved using conventional hybridoma technology and screeningagainst tumor vascular endothelial cells.

[0191] A development of the above method is that which mimics the tumorvasculature phenomenon in vitro, and where cell purification is notnecessary. In using this method, endothelial cells are subjected totumor-derived products, such as might be obtained from tumor-conditionedmedia, in cell culture rather than in an animal. This method generallyinvolves stimulating endothelial cells with tumor-conditioned medium andemploying the stimulated endothelial cells as immunogens to prepare acollection of antibodies. Again, specific antibodies should be selected,e.g., using conventional monoclonal antibody technology, or othertechniques such as combinatorial immunoglobulin phagemid librariesprepared from RNA isolated from the spleen of the immunized animal. Onewould select a specific antibody that preferentially recognizestumor-stimulated vascular endothelium and reacts more strongly withtumor-associated endothelial cells than with normal adult human tissues.

[0192] Stimulated endothelial cells contemplated to be of use in thisregard include, for example, human umbilical vein endothelial cells(HUVE), human dermal microvascular endothelial cells (HDEMC), humansaphenous vein endothelial cells, human omental fat endothelial cells,other human microvascular endothelial cells, human brain capillaryendothelial cells, and the like. It is also contemplated thatendothelial cells from another species may stimulated bytumor-conditioned media and employed as immunogens to generatehybridomas to produce an antibodies in accordance herewith, i.e., toproduce antibodies that crossreact with tumor-stimulated human vascularendothelial cells, and/or antibodies for use in pre-clinical models.

[0193] “Tumor-conditioned medium or media” are defined herein ascompositions or media, such as culture media, that contain one or moretumor-derived cytokines, lymphokines or other effector molecules. Mosttypically, tumor-conditioned medium is prepared from a culture medium inwhich selected tumor cells have been grown, and will therefore beenriched in such tumor-derived products. The type of medium is notbelieved to be particularly important, so long as it at least initiallycontains appropriate nutrients and conditions to support tumor cellgrowth. It is also, of course, possible to extract and even separatematerials from tumor-conditioned media and employ one or more of theextracted products for application to the endothelial cells.

[0194] As for the type of tumor used for the preparation of the mediumor media, one will, of course, prefer to employ tumors that mimic orresemble the tumor that will ultimately be subject to analysis ortreatment using the present invention. Thus, for example, where oneenvisions the development of a protocol for the treatment of breastcancer, one will desire to employ breast cancer cells such as ZR-75-1,T47D, SKBR3, MDA-MB-231. In the case of colorectal tumors, one maymention by way of example the HT29 carcinoma, as well as DLD-1, HCT116or even SW48 or SW122. In the case of lung tumors, one may mention byway of example NCI-H69, SW2, NCI H23, NCI H460, NCI H69, or NCI H82. Inthe case of melanoma, good examples are DX.3, A375, SKMEL-23, HMB-2,MJM, T8 or indeed VUP. In any of the above cases, it is further believedthat one may even employ cells produced from the tumor that is to betreated, i.e., cells obtained from a biopsy.

[0195] Once prepared, the tumor-conditioned media is then employed tostimulate the appearance of tumor endothelium-specific marker(s) on thecell surfaces of endothelial cells, e.g., by culturing selectedendothelial cells in the presence of the tumor-conditioned media (orproducts derived therefrom). Again, it is proposed that the type ofendothelial cell that is employed is not of critical importance, so longas it is generally representative of the endothelium associated with thevasculature of the particular tumor that is ultimately to be treated ordiagnosed. The inventors prefer to employ human umbilical veinendothelial cells (HUVE), or human dermal microvascular endothelialcells (HDMEC, Karasek, 1989), in that these cells are of human origin,respond to cytokine growth factors and angiogenic factors and arereadily obtainable. However, it is proposed that any endothelial cellthat is capable of being cultured in vitro may be employed in thepractice of the invention and nevertheless achieve beneficial results.One may mention, by way of example, cells such as EA.hy9.26, ECV304,human saphenous vein endothelial cells, and the like.

[0196] Once stimulated using the tumor-derived products, the endothelialcells are then employed as immunogens in the preparation of monoclonalantibodies (MAbs). The technique for preparing MAbs against antigeniccell surface markers is quite straightforward, and may be readilycarried out using techniques well known to those of skill in the art, asexemplified by the technique of Kohler & Milstein (1975), and furtherdescribed hereinbelow.

[0197] Generally speaking, a preferred method of preparing MAbs usingstimulated endothelial cells involves the following procedures: Cells orcell lines derived from human tumors are grown in tissue culture for ≧4days. The tissue culture supernatant (‘tumor-conditioned medium’) isremoved from the tumor cell cultures and added to cultures of HUVEC at afinal concentration of 50% (v/v). After 2 days culture the HUVEC areharvested non-enzymatically and 1-2×10⁶ cells injected intraperitoneallyinto mice. This process is repeated three times at two-weekly intervals,the final immunization being by the intravenous route. Three days laterthe spleen cells are harvested and fused with SP2/0 myeloma cells bystandard protocols (Kohler & Milstein, 1975) and hybridomas producingantibodies with the appropriate reactivity are cloned by limitingdilution.

[0198] From the resultant collection of hybridomas, one will then desireto select one of more hybridomas that produce an antibody thatrecognizes the activated vascular endothelium to a greater extent thanit recognizes non-activated vascular endothelium. One goal is theidentification of antibodies having virtually no binding affinity fornormal endothelium. However, in contrast to the prior art, in thepresent invention this property is not critical. In any event, one willgenerally identify suitable antibody-producing hybridomas by screeningusing, e.g., an ELISA, RIA, IRMA, IIF, or similar immunoassay, againstone or more types of tumor-activated endothelial cells. Once candidateshave been identified, one will desire to test for the absence ofreactivity for non-activated or “normal” endothelium or other normaltissue or cell type. In this manner, hybridomas producing antibodieshaving an undesirably high level of normal cross-reactivity for theparticular application envisioned may be excluded.

[0199] (f) Anti-Endoglin Antibodies

[0200] Using the technique described above, antibodies having relativespecificity for tumor vascular endothelium have been prepared andisolated. In one particular example, HT29 carcinoma cells were employedto prepare the conditioned medium, which was then employed to stimulateHUVE cells in culture. The resultant HT29-activated HUVE cells were thenemployed as immunogens in the preparation of a hybridoma bank, which wasELISA-screened using HT29-activated HUVE cells and by immunohistologicanalysis of sections of human tumors and normal tissues. From this bank,antibodies that recognized a tumor vascular endothelial cell antigenwere selected.

[0201] The MAbs termed tumor endothelial cell antibody 4 and tumorendothelial cell antibody 11 (TEC4 and TEC11) were obtained using theabove method. The antigen recognized by TEC4 and TEC11 was ultimatelydetermined to be the molecule endoglin. The epitopes on endoglinrecognized by TEC4 and TEC11 are present on the cell surface ofstimulated HUVE cells, and only minimally present (or immunologicallyaccessible) on the surface of non-stimulated cells. MAbs have previouslybeen raised against endoglin. However, analyzing the reactivity withHUVEC or TCM-activated HUVEC cell surface determinants by FACS orindirect immunofluorescence shows the epitopes recognized by TEC-4 andTEC-11 to be distinct from those of a previous antibody termed 44G4(Gougos & Letarte, 1988).

[0202] Although any of the known anti-endoglin antibodies (e.g., Gougos& Letarte, 1988; Gougos et al., 1992; O'Connell et al., 1992; Buhring etal., 1991) may be used in connection with the present invention, theTEC-4 and TEC-11 mAbs are envisioned to be particularly suitable. Thisis because they label capillary and venular endothelial cells moderatelyto strongly in a broad range of solid tumors (and in several chronicinflammatory conditions and fetal placenta), but display relatively weakstaining of vessels in the majority of normal, healthy adult tissues.TEC-11 is particularly preferred as it shows virtually no reactivitywith non-endothelial cells. Furthermore, both TEC-4 and TEC-11 arecomplement-fixing, which imparts to them the potential to also induceselective lysis of endothelial cells in the tumor vascular bed.

[0203] Antibodies that are cross-reactive with the MAbs TEC-4 andTEC-11, i.e., those that bind to endoglin at the same epitope as TEC-4or TEC-11, are also contemplated to be of use in this invention. Theidentification of an antibody or antibodies that bind to endoglin at thesame epitopes as TEC-4 or TEC-11 is a fairly straightforward matter.This can be readily determined using any one of variety of immunologicalscreening assays in which antibody competition can be assessed. Forexample, where the test antibodies to be examined are obtained from adifferent source to that of TEC-4 or TEC-11 , e.g., a rabbit, or areeven of a different isotype, for example, IgG1 or IgG3, a competitionELISA may be employed. In one such embodiment of a competition ELISA onewould pre-mix TEC-4 or TEC-11 with varying amounts of the testantibodies prior to applying to the antigen-coated wells in the ELISAplate. By using either anti-murine or anti-IgM secondary antibodies onewill be able to detect only the bound TEC-4 or TEC-11 antibodies—thebinding of which will be reduced by the presence of a test antibody thatrecognizes the same epitope as either TEC-4 or TEC-11.

[0204] To conduct an antibody competition study between TEC-4 or TEC-11and any test antibody, one may first label TEC-4 or TEC-11 with adetectable label, such as, e.g., biotin or an enzymatic or radioactivelabel, to enable subsequent identification. In these cases, one wouldincubate the labelled antibodies with the test antibodies to be examinedat various ratios (e.g., 1:1, 1:10 and 1:100) and, after a suitableperiod of time, one would then assay the reactivity of the labelledTEC-4 or TEC-11 antibodies and compare this with a control value inwhich no potentially competing antibody (test) was included in theincubation.

[0205] The assay may be any one of a range of immunological assays basedupon antibody binding and the TEC-4 or TEC-11 antibodies would bedetected by means of detecting their label, e.g., using streptavidin inthe case of biotinylated antibodies or by using a chromogenic substratein connection with an enzymatic label or by simply detecting theradiolabel. An antibody that binds to the same epitope as TEC-4 orTEC-11 will be able to effectively compete for binding and thus willsignificantly reduce TEC-4 or TEC-11 binding, as evidenced by areduction in labelled antibody binding. In the present case, aftermixing the labelled TEC-4 or TEC-11 antibodies with the test antibodies,suitable assays to determine the remaining reactivity include, e.g.,ELISAs, RIAs or western blots using human endoglin; immunoprecipitationof endoglin; ELISAs, RIAs or immunofluorescent staining of recombinantcells expressing human endoglin; indirect immunofluorescent staining oftumor vasculature endothelial cells; reactivity with HUVEC orTCM-activated HUVEC cell surface determinants indirectimmunofluorescence and FACS analysis. This latter method is mostpreferred and was employed to show that the epitopes recognized by TEC-4and TEC-11 are distinct from that of 44G4 (Gougos & Letarte, 1988).

[0206] The reactivity of the labelled TEC-4 or TEC-11 antibodies in theabsence of any test antibody is the control high value. The control lowvalue is obtained by incubating the labelled antibodies with unlabelledantibodies of the same type, when competition would occur and reducebinding of the labelled antibodies. A significant reduction in labelledantibody reactivity in the presence of a test antibody is indicative ofa test antibody that recognizes the same epitope, i.e., one that“cross-reacts” with the labelled antibody. A “significant reduction” inthis aspect of the present application may be defined as a reproducible(i.e., consistently observed) reduction in binding of at least about10-50% at a ratio of about 1:1, or more preferably, of equal to orgreater than about 90% at a ratio of about 1:100.

[0207] The use of “cross-reactivity assays”, as described above in thecontext of TEC-4 and TEC-11 antibodies, may be applied to any antibodyfor use in the present invention. Therefore, antibodies that bind to acomponent of a tumor cell, a component of tumor vasculature, a tumorcell-associated component, a tumor vasculature-associated component, atumor extracellular matrix component, or to any cell type listed herein,at the same epitope as any of the antibodies listed herein, asdetermined by an antibody competition assay, will be an antibody thatfalls under the scope of this invention when combined with a coagulatingagent to form a bispecific ligand.

[0208] (g) Use of Vascular Endothelial Cell Binding Ligands

[0209] Biological ligands that are known to bind or interact withendothelial cell surface molecules, such as growth factor receptors, mayalso be employed as a targeting component.

[0210] The growth factors or ligands contemplated to be useful astargets in this sense include VEGF/VPF, FGF, TGFβ, ligands that bind toa TIE, tumor-associated fibronectin isoforms, scatter factor, hepatocytegrowth factor (HGF), platelet factor 4 (PF4), PDGF and TIMP.

[0211] Particularly preferred targets are VEGF/VPF, the FGF family ofproteins and TGFβ. Abraham et al. (1986) cloned FGF, which is thereforeavailable as a recombinant protein. As reported by Ferrara et al.(1991), four species of VEGF having 121, 165, 189, and 206 amino acidshave been cloned.

[0212] (h) Targeting of Bound Ligands

[0213] Antibodies or specific targeting ligands may also be directed toany component that binds to the surface of vascular endothelial cells ina disease site, such as a tumor. Such components are exemplified bytumor-derived ligands and antigens, such as growth factors, that bind tospecific cell surface receptors already present on the endothelialcells, or to receptors that have been induced, or over-expressed, onsuch cells in response to the tumor environment. Tumorvasculature-associated targets may also be termed tumor-derivedendothelial cell binding factors.

[0214] A level of specificity required for successful disease targetingwill be achieved partly because the local endothelial cells will beinduced to express, or reveal, receptors that are not present, or areunder-expressed or masked, on normal endothelial cells. With tumors,further specificity will result due to the fact that endothelial cellsin the tumor will capture the tumor-derived factors, and bind them tothe cell surface, reducing the amount of ligand available for othertissues. When combined with the further dilution of the factor or ligandby distribution in the blood and tissue fluid pool, endothelial cells innormal tissues will be expected to bind relatively little of suchfactors. Thus, operationally, cell-surface bound ligands or factors willbe able to used as a tumor endothelial cell marker.

[0215] In addition to manufacture by the tumor cells themselves, tumorendothelial cell binding factors may also originate from other celltypes, such as macrophages and mast cells, that have infiltrated tumors,or may be elaborated by platelets that become activated within thetumor.

[0216] Further growth factors or ligands contemplated to be useful astumor vasculature-associated targets include EGF, FGF, VEGF, TGFβ, HGF(NaKamura, 1991), angiotropin, TGF-α, TNF-α, PD-ECGF and TIE bindingligands (Bicknell and Harris, 1992). The currently preferred targets areVEGF/VPF, the FGF family of proteins, transforming growth factor-β(TGF-β); TGF-α; tumor necrosis factor-α (TNF-α); angiotropin;platelet-derived endothelial cell growth factor (PD-ECGF); TIE bindingligands; pleiotropin.

[0217] Another aspect of the present invention is the use of targetingantibodies, or binding regions therefrom, that are specific for epitopespresent only on ligand-receptor complexes, which epitopes are absentfrom both the individual (free) ligand and the receptor in its unboundform. These antibodies recognize and bind to the unique conformationthat results when a ligand, such as a growth factor, binds to itsreceptor, such as a growth factor receptor, to form a specifically boundcomplex. Such epitopes are not present on the uncomplexed forms of theligands or receptors.

[0218] The inventors contemplate that the ligand-receptor complexes towhich these antibodies bind are present in significantly higher numberon tumor-associated endothelial cells than on non-tumor associatedendothelial cells. Such antibodies will therefore be useful as targetingagents and will serve to further increase the specificity of thebispecific coagulants of the invention.

[0219] (i) Receptor Constructs

[0220] Soluble binding domains of endothelial cell surface receptors arealso contemplated for use as targeting ligands in the present invention.This concept is generally based upon the well-known sandwich bindingphenomena that has been exploited in a variety of in vitro and in vivobinding protocols. Basically, as the endothelial cells express specificreceptors, the cells bind to and adsorb the corresponding ligands, theligands are then available for binding to further receptor constructsshould they be introduced into the system.

[0221] A range of useful endothelial cell receptors has been identifiedin the foregoing sections, with VEGF/VPF, FGF, TGFβ, TIE-1 and TIE-2being particularly preferred targets. Each of these receptors could bemanipulated to form a soluble binding domain for use as a targetingligand.

[0222] 4. Disease-Associated Stromal Cell Targets

[0223] (a) Extracellular Matrix/Stromal Targets

[0224] The usefulness of the basement membrane markers in tumoralpathology was described by Birembaut et al. (1985). These studies showedthat the distribution of basement membrane (BM) markers, type IVcollagen, laminin (LM), heparan sulphate proteoglycan (HSP) andfibronectin (FN) were disrupted in tumoral pathology. Burtin et. al.(1983) also described alterations of the basement membrane andconnective tissue antigens in human metastatic lymph nodes.

[0225] A preferred target for use with the invention is RIBS. Ugarova etal. (1993) reported that conformational changes occur in fibrinogen andare elicited by its interaction with the platelet membrane glycoproteinGPIIb-IIIa. The binding of fibrinogen to membrane glycoproteinGPIIb-IIIa on activated platelets leads to platelet aggregation. Thisinteraction results in conformational changes in fibrinogen as evidencedby the expression of receptor-induced binding sites, RIBS:, epitopeswhich are expressed by the bound but not the free ligand.

[0226] Two RIBS epitopes have been localized by Ugarova et al. (1993).One sequence resides at γ112-119 and is recognized by MAb 9F9; thesecond is the RGDF sequence at Aα 95-98 and is recognized by mAb 155B16.These epitopes are also exposed by adsorption of fibrinogen onto aplastic surface and digestion of the molecule by plasmin. Proteolyticexposure of the epitopes coincides with cleavage of thecarboxyl-terminal aspects of the Aα-chains to form fragment x₂. Theinaccessibility of the RGDF sequence at Aα 95-98 in fibrinogen suggeststhat this sequence does not participate in the initial binding of themolecule to GPIIb-IIIa.

[0227] Binding of fibrinogen to its receptor alters the conformation ofthe carboxyl-terminal aspects of the Aα-chains, exposing the sequenceswhich reside in the coiled-coil connector segments between the D and Edomains of the molecule, generating the RIBS epitopes. In practicalterms, the RIBS sequences are proposed as epitopes for use in targetingwith a coaguligand. The MAbs 9F9 and 155B16 may thus be advantageouslyused, as may the antibodies described by Zamarron et al. (1991).

[0228] (b) Additional Cellular Targets

[0229] The present invention has the further advantage that it may beused to direct coagulants to disease-associated vasculature by targetingthem to cell types found within the disease region.

[0230] Platelets participate in hemostasis and thrombosis by adhering toinjured blood vessel walls and accumulating at the site of injury.Although platelet deposition at sites of blood vessel injury isresponsible for the primary arrest of bleeding under physiologicconditions, it can lead to vascular occlusion with ensuing ischemictissue damage and thrombus embolization under pathologic conditions.

[0231] Interactions of platelets with their environment and with eachother represent complex processes that are initiated at the cellsurface. The surface membrane, therefore, provides a reactive interfacebetween the external medium, including components of the blood vesselwall and plasma, and the platelet interior.

[0232] p-155, a multimeric platelet protein that is expressed onactivated platelets (Hayward et al., 1991), may be targeted using theinvention. Platelets respond to a large number of stimuli by undergoingcomplex biochemical and morphological changes. These changes areinvolved in physiological processes including adhesion, aggregation, andcoagulation. Platelet activation produces membrane alterations that canbe recognized by monoclonal antibodies. The monoclonal antibody JS-1(Hayward et al., 1991) is one such antibody contemplated for use as partof a coaguligand.

[0233] Ligand-induced binding sites (LIBS) are sites expressed on cellsurface receptors only after ligand binding causes the receptor tochange shape, mediate subsequent biological events. These may be seen ascounterparts to RIBS and are also preferred targets for use with thepresent invention.

[0234] 13 anti-LIBS antibodies have been developed by Frelinger et. al.(1990; 1991), any one of which may be used to deliver a coagulant to adisease or tumor site in accordance herewith. The murine monoclonalantiplatelet antibodies MA-TSPI-1 (directed against humanthrombospondin) and MA-PMI-2, MA-PMI-1, and MA-LIBS-1 (directed againstLIBS on human platelet glycoprotein IIb/IIIa) of Dewerchin et al. (1991)may also be used, as may RUU 2.41 and LIBS-1 of Heynen et al. (1994);OP-G2 of Tomiyama et al. (1992); and Ab-15.

[0235] Many other targets, such as antigens on smooth muscle cells,pericytes, fibroblasts, macrophages and infiltrating lymphocytes andleukocytes may also be used.

[0236] B. Coagulating Agents

[0237] The second arm or element of the bispecific agents of theinvention will be a component that is capable of promoting coagulation.“Coagulation promoting agents” may be coagulation factors, factors thatindirectly stimulate coagulation, or they may be in the form of a secondbinding region that is capable of binding and releasing a coagulationfactor or factor that indirectly stimulates coagulation.

[0238] 1. Coagulation Factors

[0239] A variety of coagulation factors may be used in connection withthe present invention, as exemplified by the agents set forth below.Where a coagulation factor is covalently linked to a first bindingagent, a site distinct from its functional coagulating site is used tojoin the molecules. Appropriate joining regions distinct from the activesites, or functional regions, of the coagulation factors are alsodescribed in each of the following sections.

[0240] (a) Tissue Factor

[0241] Tissue factor (TF) is one agent capable of initiating bloodcoagulation. TF is the activator of the extrinsic pathway of bloodcoagulation and is not in direct contact with the blood underphysiologically normal conditions (Osterud et al., 1986; Nemerson, 1988;Broze, 1992; Ruf & Edington, 1994). In vascular damage or activation bycertain cytokines or endotoxin, however, TF will be exposed to theblood, either by the (sub) endothelial. cells (Weiss et al., 1989) or bycertain blood cells (Warr et al., 1990). TF will then complex withfactor VIIa, which under normal conditions circulates at lowconcentrations in the blood (Wildgoose et al., 1992), and the TF/factorVIIa complex will start the coagulation cascade through the activationof factor X into factor Xa. The cascade will ultimately result in theformation of fibrin.

[0242] For this sequence of events to occur, the TF:VIIa complex has tobe associated with a phospholipid surface upon which thecoagulation-initiation complexes with factors IX or X can assemble (Rufet al., 1991; Paborsky et al., 1991; Bach et al., 1986). For thisreason, truncated TF (or tTF), from which the transmembrane andcytoplasmic regions have been removed by truncating the gene, is asoluble protein having one hundred-thousandth of the factor X-activatingactivity of native TF (Ruf et al., 1991).

[0243] (b) Clotting Factors

[0244] Thrombin, Factor V/Va and derivatives, Factor VIII/VIIIa andderivatives, Factor IX/IXa and derivatives, Factor X/Xa and derivatives,Factor XI/XIa and derivatives, Factor XII/XIIa and derivatives, FactorXIII/XIIIa and derivatives, Factor X activator and Factor V activatormay also be used in the present invention.

[0245] (c) Venom Coagulants

[0246] Russell's viper venom was shown to contain a coagulant protein byWilliams and Esnouf in 1962. Kisiel (1979) isolated a venom glycoproteinthat activates Factor V; and Di Scipio et al. (1977) showed that aprotease from the venom activates human Factor X. The Factor X activatoris the component contemplated for use in this invention.

[0247] Monoclonal antibodies specific for the Factor X activator presentin Russell's viper venom have also been produced (e.g., MP1 ofPukrittayakamee et al., 1983), and could be used to deliver the agent toa specific target site within the body.

[0248] (d) Prostaglandins and Synthetic Enzymes

[0249] Thromboxane A₂ is formed from endoperoxides by the sequentialactions of the enzymes cyclooxygenase and thromboxane synthetase inplatelet microsomes. Thromboxane A₂ is readily generated by plateletsand is a potent vasoconstrictor, by virtue of its capacity to produceplatelet aggregation (Whittle et al., 1981).

[0250] Both thromboxane A₂ and active analogues thereof are contemplatedfor use in the present invention. A synthetic protocol for generatingthromboxane A₂ is described by Bhagwat et al. (1985). The thromboxane A₂analogues described by Ohuchida et. al. (1981) (especially compound 2)are particularly contemplated for use herewith.

[0251] It is possible that thromboxane synthase, and other enzymes thatsynthesize platelet-activating prostaglandins, may also be used as“coagulants” in the present context. Shen and Tai (1986a;b) describemonoclonal antibodies to, and immunoaffinity purification of,thromboxane synthase; and Wang et. al. (1991) report the cDNA for humanthromboxane synthase.

[0252] (e) Inhibitors of Fibrinolysis

[0253] α2-antiplasmin, or α2-plasmin inhibitor, is a proteinaseinhibitor naturally present in human plasma that functions toefficiently inhibit the lysis of fibrin clots induced by plasminogenactivator (Moroi & Aoki, 1976). α2-antiplasmin is a particularly potentinhibitor, and is contemplated for use in the present invention.

[0254] α2-antiplasmin may be purified as first described by Moroi andAoki (1976). Other purification schemes are also available, such asusing affinity chromatography on plasminogen-Sepharose, ion-exchangechromatography on DEAE-Sephadex and chromatography onConcanavalin-A-Sepharose; or using affinity chromatography on aSepharose column bearing an elastase-digested plasminogen formulationcontaining the three N-terminal triple-loop structures in the plasminA-chain (LBSI), followed by gel filtration (Wiman & Collen, 1977; Wiman,1980, respectively).

[0255] As the cDNA sequence for α2-antiplasmin is available (Tone etal., 1977), a preferred method for α2-antiplasmin production will be viarecombinant expression.

[0256] Monoclonal antibodies against α2-antiplasmin are also availablethat may be used in the bispecific binding ligand embodiments of theinvention. For example, Hattey et al. (1987) described two MAbs againstα2-antiplasmin, MPW2AP and MPW3AP. As each of these MAbs were reportedto react equally well with native α2-antiplasmin, they could both beused to deliver exogenous α2-antiplasmin to a target site or to garnerendogenous α2-antiplasmin and concentrate it within the targeted region.Other antibodies, such as JTPI-2, described by Mimuro and colleagues,could also be used.

[0257] 2. Agents that Bind Coagulation Factors

[0258] Another group of bispecific coagulating ligands of this inventionare those in which the targeting region is not directly linked to acoagulation factor, but is linked to a second binding region that bindsto a coagulating factor.

[0259] Where a second binding region is used to bind and deliver acoagulation factor, the binding region is chosen so that it recognizes asite on the coagulation factor that does not significantly impair itsability to induce coagulation. The regions of the coagulation factorssuitable for binding in this manner will generally be the same as thoseregions that are suitable for covalent linking to the targeting region,as described in the previous sections.

[0260] However, in that bispecific ligands of this class may be expectedto release the coagulation factor following delivery to the tumor siteor region, there is more flexibility allowed in the regions of thecoagulation factor suitable for binding to a second binding agent orantibody. Another advantage is that bispecific antibodies can bepre-localized before infusion of tTF which may reduce the amount of tTfrequired and hence toxicity.

[0261] Suitable second binding regions for use in this manner, willgenerally be antigen combining sites of antibodies that have bindingspecificity for the coagulation factor, including functional portions ofantibodies, such as scFv, Fv, Fab′, Fab and F(ab′)₂ fragments.

[0262] Bispecific binding ligands that contain antibodies, or fragmentsthereof, directed against Tissue Factor, Thrombin, Prekallikein, FactorV/Va, Factor VIII/VIIIa, Factor IX/IXa, Factor X/Xa, Factor XI/XIa,Factor XII/XIIa, Factor XIII/XIIIa, Russell's viper venom, thromboxaneA₂ or α2-antiplasmin are exemplary embodiments of this aspect of theinvention.

[0263] C. Linkage Means

[0264] The first, targeting region and second, coagulating region willbe operatively linked to allow each region to perform its intendedfunction without significant impairment. Thus, the targeting region iscapable of binding to the intended target, as selected from the range oftumor environment targets, and the coagulating region is capable ofdirectly or indirectly, e.g., through the release of a bound factor,promoting blood coagulation or clotting.

[0265] To assess the targeting region binding function, all that isrequired is to conduct a binding assay to ensure that the bispecificligand still binds to the targeted component in substantially the samemanner as the uncomplexed first binding region. The suitable bindingassays are of the type usually seen in immunological binding assays,where the first targeting region is an antibody, and/or otherbiochemical binding assays, e.g., those using ¹²⁵iodine labeled proteinsor other radiolabeled components, as used to assess ligand-receptorbinding, to generate Scatchard plots, and the like.

[0266] The target antigen or component in such assays may be provided inmany forms, including proteins purified from natural or recombinantsources, membrane enriched preparations, intact cells and tissuesections. Generally, where protein compositions are used, they willimmobilized on a solid support, such as a microtitre plate, a membrane,or even on a column matrix. It is also generally preferred to use atarget composition that reflects the physiological target, therefore asthe target will usually be cell-associated, the use of compositions thatinclude intact cells, including tissues and the cells themselves, isalso preferred.

[0267] The various immunological assays available to confirm thefunctional binding of a bispecific complex include, e.g., Western blots,ELISAS, ELISAs using fixed cells, immunohistochemistry, and fluorescentactivated cell sorting (FACS). The execution of all such assays isgenerally known to those of skill in the art, and is further disclosedherein.

[0268] Assessing the targeting region binding function of a bispecificcompound in any of the above or other binding assays is astraightforward matter, where the bispecific ligand and the uncomplexedfirst binding region will most usually be run in a parallel assay, underthe same conditions, to enable ready comparison. Effective bispecificligands will bind to the target without significant impairment, i.e., insubstantially the same manner as the uncomplexed first binding region.Taking the uncomplexed binding region assay result as the 100% referencevalue, “substantial binding” of the bispecific ligand, as used herein,means that the bispecific ligand exhibits at least about 50% binding,and more preferably, between about 50% and about 80% binding, and mostpreferably, between about 80% and about 100% binding.

[0269] Where the bispecific ligand includes a second binding region thatbinds to a coagulant, e.g., it is a bispecific antibody, further usefulassays are those of the type that allow the binding functions of botharms of the bispecific ligand to be assessed at the same time. Forexample, this may be achieved by assessing the binding of a radiolabeledcoagulant to a target cell via bridging with the bispecific ligand orantibody. Such an assay is exemplified by the binding of tTF to targetcells using the B21-2/10H10 bispecific antibody, as described in ExampleII.

[0270] Determining the coagulating agent function of the bispecificligand is also a straightforward matter. All that is required here is toconduct a coagulation assay using the bispecific ligand and ensure thatit functions to promote coagulation in substantially the same manner asthe uncomplexed coagulating agent. This is true for “coagulating agents”that are both coagulation factors themselves and those that are secondbinding regions that bind to a coagulation factor. Naturally, in thelatter case, in an in vitro or ex vivo assay, the bispecific ligand willbe precomplexed with the coagulation factor to allow binding to thesecond binding region.

[0271] One suitable coagulation assay is that in which the bispecificligands, pre-complexed with coagulant if necessary, are admixed with aplasma sample. The appearance of fibrin strands is indicative ofcoagulation in this assay. Effective bispecific ligands would thus beexpected to reduce the time taken for fibrin strands to appear, andparticularly, to significantly reduce the elapsed time in comparison tocontrol levels.

[0272] A variation of the above assay involves first exposingappropriate target cells to the bispecific ligand under conditionseffective, and for a time sufficient, to allow binding, washing thecells to remove non-specifically bound components and then resuspendingthe washed cells in plasma. Only cells effectively coated with thebispecific ligand would be expected to reduce the time taken for fibrinstrands to appear in the assay. This type of assay is preferred in thatit is, in itself, an assay that assesses both of the functions of thebispecific construct, i.e., initial targeting to the cell and subsequentlocalized coagulation.

[0273] To compare the coagulating function of a bispecific compound tothat of an uncomplexed coagulating agent, parallel assays may again beconducted. Effective bispecific ligands will function to promotecoagulation without significant impairment, i.e., will function insubstantially the same manner as the uncomplexed coagulating agent.Taking the uncomplexed coagulant assay result as the 100% referencevalue, “substantial function”, as used herein, means that the bispecificligand exhibits at least about 50% coagulation, and more preferably,between about 50% and about 80% coagulation, and most preferably,between about 80% and about 100% coagulation.

[0274] The two functional regions of the bispecific ligands may bejoined using synthetic chemistry techniques or recombinant DNAtechniques. Each of these techniques are routinely employed and wellknown to those of skill in the art, and are further exemplified inExample I and by the details set forth below.

[0275] 1. Biochemical Cross-Linkers

[0276] The joining of an antibody, or other targeting component, to acoagulating agent will generally employ the same technology as developedfor the preparation of immunotoxins. However, considerable advantagesare apparent in the present technology, as the consequences of a certainamount of uncomplexed coagulating agent becoming availablephysiologically are not contemplated to be particularly severe. Thus,the stability requirements for any cross-linkers are not so stringent asfor linkers employed in other constructs, such as immunotoxins.Therefore, it can be considered as a general guideline that anybiochemical crosslinker that is appropriate for use in an immunotoxinwill also be of use in the present context, and additional linkers mayalso be considered.

[0277] In addition to toxins, a variety of other chemotherapeutic andpharmacological agents have been linked to antibodies to form conjugatesthat have been shown to function pharmacologically (see, e.g., Vaickuset al., 1991). Exemplary antineoplastic agents that have beeninvestigated include doxorubicin, daunomycin, methotrexate andvinblastine, amongst others (Dillman et al., 1988; Pietersz et al.,1988). Moreover, the attachment of other agents such as neocarzinostatin(Kimura et al., 1983), macromycin (Manabe et al., 1984), trenimon(Ghose, 1982) and α-amanitin (Davis & Preston, 1981) has been described.The linking technology described in each of the foregoing scientificpapers is also contemplated for use in connection with the presentinvention.

[0278] Cross-linking reagents are used to form molecular bridges thattie together functional groups of two different molecules, e.g., abinding and coagulating agent. To link two different proteins in astep-wise manner, heterobifunctional cross-linkers can be used thateliminate unwanted homopolymer formation. TABLE VI HETEROBIFUNCTIONALCROSS-LINKERS Spacer Arm Length\after linker Reactive Toward Advantagesand Applications cross-linking SMPT Primary amines Greater stability11.2 Å Sulfhydryls SPDP Primary amines Thiolation  6.8 Å SulfhydrylsCleavable cross-linking LC-SPDP Primary amines Extended spacer arm 15.6Å Sulfhydryls Sulfo-LC-SPDP Primary amines Extended spacer arm 15.6 ÅSulfhydryls Water-soluble SMCC Primary amines Stable maleimide reactivegroup 11.6 Å Sulfhydryls Enzyme-antibody conjugation Hapten-carrierprotein conjugation Sulfo-SMCC Primary amines Stable maleimide reactivegroup 11.6 Å Sulfhydryls Water-soluble Enzyme-antibody conjugation MBSPrimary amines Enzyme-antibody conjugation  9.9 Å SulfhydrylsHapten-carrier protein conjugation Sulfo-MBS Primary aminesWater-soluble  9.9 Å Sulfhydryls SIAB Primary amines Enzyme-antibodyconjugation 10.6 Å Sulfhydryls Sulfo-SIAB Primary amines Water-soluble10.6 Å Sulfhydryls SMPB Primary amines Extended spacer arm 14.5 ÅSulfhydryls Enzyme-antibody conjugation Sulfo-SMPB Primary aminesExtended spacer arm 14.5 Å Sulfhydryls Water-soluble EDC/Sulfo-NHSPrimary amines Hapten-Carrier conjugation 0 Carboxyl groups ABHCarbohydrates Reacts with sugar groups 11.9 Å Nonselective

[0279] An exemplary heterobifunctional cross-linker contains tworeactive groups: one reacting with primary amine group (e.g., N-hydroxysuccinimide) and the other reacting with a thiol group (e.g., pyridyldisulfide, maleimides, halogens, etc.). Through the primary aminereactive group, the cross-linker may react with the lysine residue(s) ofone protein (e.g., the selected antibody or fragment) and through thethiol reactive group, the cross-linker, already tied up to the firstprotein, reacts with the cysteine residue (free sulfhydryl group) of theother protein (e.g., the coagulant).

[0280] It can therefore be seen that the preferred coagulants orcoagulant binding regions will generally have, or be derivatized tohave, a functional group available for cross-linking purposes. Thisrequirement is not considered to be limiting in that a wide variety ofgroups can be used in this manner. For example, primary or secondaryamine groups, hydrazide or hydrazine groups, carboxyl alcohol,phosphate, or alkylating groups may be used for binding orcross-linking. For a general overview of linking technology, one maywish to refer to Ghose & Blair (1987).

[0281] The spacer arm between the two reactive groups of a cross-linkersmay have various length and chemical compositions. A longer spacer armallows a better flexibility of the conjugate components while someparticular components in the bridge (e.g., benzene group) may lend extrastability to the reactive group or an increased resistance of thechemical link to the action of various aspects (e.g., disulfide bondresistant to reducing agents). The use of peptide spacers, such asL-Leu-L-Ala-L-Leu-L-Ala, is also contemplated.

[0282] It is preferred that a cross-linker having reasonable stabilityin blood will be employed. Numerous types of disulfide-bond containinglinkers are known that can be successfully employed to conjugatetargeting and coagulating agents. Linkers that contain a disulfide bondthat is sterically hindered may prove to give greater stability in vivo,preventing release of the coagulant prior to binding at the site ofaction. These linkers are thus one preferred group of linking agents.

[0283] One of the most preferred cross-linking reagents for use inimmunotoxins is SMPT, which is a bifunctional cross-linker containing adisulfide bond that is “sterically hindered” by an adjacent benzene ringand methyl groups. It is believed that stearic hindrance of thedisulfide bond serves a function of protecting the bond from attack bythiolate anions such as glutathione which can be present in tissues andblood, and thereby help in preventing decoupling of the conjugate priorto the delivery of the attached agent to the tumor site. It iscontemplated that the SMPT agent may also be used in connection with thebispecific coagulating ligands of this invention.

[0284] The SMPT cross-linking reagent, as with many other knowncross-linking reagents, lends the ability to cross-link functionalgroups such as the SH of cysteine or primary amines (e.g., the epsilonamino group of lysine). Another possible type of cross-linker includesthe heterobifunctional photoreactive phenylazides containing a cleavabledisulfide bond such as sulfosuccinimidyl-2-(p-azido salicylamido)ethyl-1,3′-dithiopropionate. The N-hydroxy-succinimidyl group reactswith primary amino groups and the phenylazide (upon photolysis) reactsnon-selectively with any amino acid residue.

[0285] In addition to hindered cross-linkers, non-hindered linkers canalso be employed in accordance herewith. Other useful cross-linkers, notconsidered to contain or generate a protected disulfide, include SATA,SPDP and 2-iminothiolane (Wawrzynczak & Thorpe, 1987). The use of suchcross-linkers is well understood in the art.

[0286] Once conjugated, the bispecific agent will generally be purifiedto separate the conjugate from unconjugated targeting agents orcoagulants and from other contaminants. It is important to removeunconjugated targeting agent to avoid the possibility of competition forthe antigen between conjugated and unconjugated species. A large anumber of purification techniques are available for use in providingconjugates of a sufficient degree of purity to render them clinicallyuseful. Purification methods based upon size separation, such as gelfiltration, gel permeation or high performance liquid chromatography,will generally be of most use. Other chromatographic techniques, such asBlue-Sepharose separation, may also be used.

[0287] 2. Recombinant Fusion Proteins

[0288] The bispecific targeted coagulants of the invention may also befusion proteins prepared by molecular biological techniques. The use ofrecombinant DNA techniques to achieve such ends is now standard practiceto those of skill in the art. These methods include, for example, invitro recombinant DNA techniques, synthetic techniques and in vivorecombination/genetic recombination. DNA and RNA synthesis may,additionally, be performed using an automated synthesizers (see, forexample, the techniques described in Sambrook et al., 1989; and Ausubelet al., 1989).

[0289] In general, to prepare a fusion a protein one would join a DNAcoding region, such as a gene or cDNA, encoding a binding ligand orother targeting region to a DNA coding region (i.e., gene or cDNA)encoding a coagulation factor or coagulant binding region. Thistypically involves preparing an expression vector that comprises, in thesame reading frame, a first DNA segment encoding the first bindingregion operatively linked to a second DNA segment encoding thecoagulation factor. The sequences are attached in a manner such thattranslation of the total nucleic acid yields the desired bispecificcompounds of the invention. Expression vectors contain one or morepromoters upstream of the inserted DNA regions that act to promotetranscription of the DNA and to thus promote expression of the encodedrecombinant protein. This is the meaning of “recombinant expression”.

[0290] Should a particular binding region or coagulant be preferred, andthe encoding DNA not instantly available, it may be obtained using thetechniques of “molecular cloning” in which a DNA molecule encoding thedesired protein is obtained from a DNA library (e.g., a cDNA or genomiclibrary). In such procedures, an appropriate DNA library is screened,e.g., using an expression screening protocol employing antibodiesdirected against the protein, or activity assays. Alternatively,screening may be based on the hybridization of oligonucleotide probes,designed from a consideration of portions of the amino acid sequence ofthe protein, or from the DNA sequences of genes encoding relatedproteins. The operation of such screening protocols are well known tothose of skill in the art and are described in detail in the scientificliterature, for example, in Sambrook et al. (1989).

[0291] When produced via recombinant DNA techniques, the targetingagent/coagulating agent compounds of the invention are referred to as“fusion proteins”. It is to be understood that such fusion proteinscontain, at least, a targeting agent and a coagulating agent as definedin this invention, and that the agents are operatively attached. Thefusion proteins may also include additional peptide sequences, such aspeptide spacers which operatively attach the targeting agent andcoagulating agent compounds, as long as such additional sequences do notappreciably affect the targeting or coagulating activities of theresultant fusion protein.

[0292] It will be understood that the recombinant bispecific proteinligands may differ from those bispecific constructs generated bychemically cross-linking the so-called naturally-produced proteins. Inparticular, the degree of post-translational modifications, such as, forexample, glycosylation and phosphorylation may be different betweenrecombinant fusions and chemical fusions of the same two proteins. Thisis not contemplated to be a significant problem, however, those of skillin the art will know to confirm that a recombinant fusion proteinfunctions as intended, and expected from other data, before use in aclinical setting.

[0293] One advantage of recombinant expression is that the linkingregions can be readily manipulated so that, e.g., their length and/oramino acid composition is readily variable. Non-cleavable peptidespacers may be provided to operatively attach the two agents of theinvention, if desired. Equally, peptides with unique cleavage sitescould be inserted between the two components.

[0294] If desired in a specific instance, it is possible to provide apeptide spacer operatively attaching the targeting agent and coagulatingagent which is capable of folding into a disulfide-bonded loopstructure. Proteolytic cleavage within the loop would then yield aheterodimeric polypeptide wherein the targeting agent and thecoagulating agent are linked by only a single disulfide bond (see, forexample, Lord et al., 1992).

[0295] Many standard techniques are available to construct expressionvectors containing the appropriate nucleic acids andtranscriptional/translational control sequences in order to achieveprotein expression in a variety of host-expression systems. The celltypes available for expression include, but are not limited to,microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformedwith recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expressionvectors containing targeting agent/coagulant coding sequences; yeast(e.g., Saccharomyces, Pichia) transformed with recombinant yeastexpression vectors containing targeting agent/coagulating agent codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing the targetingagent/coagulating agent coding sequences; plant cell systems infectedwith recombinant virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinantplasmid expression vectors (e.g., Ti plasmid) containing the targetingagent/coagulant coding sequence; and mammalian cell systems (e.g., COS,CHO, BHK, 293, 3T3) harboring recombinant expression constructscontaining promoters derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g., theadenovirus late promoter; the vaccinia virus 7.5K promoter).

[0296] In bacterial systems a number of expression vectors may beadvantageously selected depending upon the use intended for thetargeting agent/coagulating agent construct being expressed. Forexample, when large quantities of bispecific agent are to be produced,vectors that direct the expression of high levels of fusion proteinproducts that are readily purified may be desirable. Such vectorsinclude, but are not limited to, the E. coli expression vector pUR278(Ruther et al., 1983), in which the targeting agent/coagulating agentcoding sequence may be ligated individually into the vector in framewith the lac Z coding region so that a fusion protein additionallycontaining a portion of the lac Z product is provided; pIN vectors(Inouye et al., 1985; Van Heeke et al., 1989); and the like. pGEXvectors may also be used to express foreign polypeptides, such as thetargeting agent/coagulating agent combinations as fusion proteinsadditionally containing glutathione S-transferase (GST). In general,such fusion proteins are soluble and can easily be purified from lysedcells by adsorption to glutathione-agarose beads followed by elution inthe presence of free glutathione. The pGEX vectors are designed toinclude thrombin or factor Xa protease cleavage sites so that thebinding agent/coagulant protein of the overall fusion protein can bereleased from the GST moiety.

[0297] In a useful insect system, Autograph californica nuclearpolyhidrosis virus (AcNPV) is used as a vector to express foreign genes.The virus grows in Spodoptera frugiperda cells. The targetingagent/coagulating agent coding sequences may be cloned intonon-essential regions (for example the polyhedrin gene) of the virus andplaced under control of an AcNPV promoter (for example the polyhedrinpromoter). Successful insertion of the bispecific ligand codingsequences will result in inactivation of the polyhedrin gene andproduction of non-occluded recombinant virus (i.e., virus lacking theproteinaceous coat coded for by the polyhedrin gene). These recombinantviruses are then used to infect Spodoptera frugiperda cells in which theinserted gene is expressed (e.g., see Smith et al., 1983; *U.S. Pat. No.4,215,051, Smith).

[0298] In mammalian host cells, a number of viral based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, the targeting agent/coagulating agent codingsequences may be ligated to an adenovirus transcription/translationcontrol complex, e.g., the late promoter and tripartite leader sequence.This chimeric gene may then be inserted in the adenovirus genome by invitro or in vivo recombination. Insertion in a non-essential region ofthe viral genome (e.g., region E1 or E3) will result in a recombinantvirus that is viable and capable of expressing bispecific proteins ininfected hosts (e.g., see Logan et al., 1984).

[0299] Specific initiation signals may also be required for efficienttranslation of inserted targeting agent/coagulating agent codingsequences. These signals include the ATG initiation codon and adjacentsequences. Exogenous translational control signals, including the ATGinitiation codon, may additionally need to be provided. One of ordinaryskill in the art would readily be capable of determining this andproviding the necessary signals. It is well known that the initiationcodon must be in phase (or in-frame) with the reading frame of thedesired coding sequence to ensure translation of the entire insert.These exogenous translational control signals and initiation codons canbe of a variety of origins, both natural and synthetic. The efficiencyof expression may be enhanced by the inclusion of appropriatetranscription enhancer elements, transcription terminators, etc. (seeBittner et al., 1987).

[0300] In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins. Appropriate cells lines or hostsystems can be chosen to ensure the correct modification and processingof the foreign protein expressed. To this end, eukaryotic host cellswhich possess the cellular machinery for proper processing of theprimary transcript, glycosylation, and phosphorylation of the geneproduct may be used. Such mammalian host cells include, but are notlimited to, CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, etc.

[0301] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines that stablyexpress constructs encoding the targeting agent/coagulant ligands may beengineered. Rather than using expression vectors that contain viralorigins of replication, host cells can be transformed with targetingagent/coagulant DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines.

[0302] A number of selection systems may be used, including, but notlimited, to the herpes simplex virus thymidine kinase (Wigler et al.,1977), hypoxanthine-guanine phosphoribosyltransferase (Szybalska et al.,1962), and adenine phosphoribosyltransferase genes (Lowy et al., 1980)can be employed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection fordhfr, which confers resistance to methotrexate (Wigler et al., 1980;O'Hare et al., 1981); gpt, which confers resistance to mycophenolic acid(Mulligan et al., 1981); neo, which confers resistance to theaminoglycoside G-418 (Colberre-Garapin et al., 1981); and hygro, whichconfers resistance to hygromycin (Santerre et al., 1984).

[0303] D. Antibodies

[0304] Where antibodies are used as one or both portions of thebispecific ligand, the choice of antibody will generally be dependent onthe type tumor and coagulating ligand chosen. However, certainadvantages may be achieved through the application of particular typesof antibodies. For example, while IgG based antibodies may be expectedto exhibit better binding capability and slower blood clearance thantheir Fab′ counterparts, Fab′ fragment-based compositions will generallyexhibit better tissue penetrating capability.

[0305] 1. Monoclonal Antibodies

[0306] Means for preparing and characterizing antibodies are well knownin the art (See, e.g., *Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, 1988).

[0307] The methods for generating monoclonal antibodies (MAbs) generallybegin along the same lines as those for preparing polyclonal antibodies.Briefly, a polyclonal antibody is prepared by immunizing an animal withan immunogenic composition in accordance with the present invention,either with or without prior immunotolerizing, depending on the antigencomposition and protocol being employed (e.g., tolerizing to a normalcell population and then immunizing with a tumor cell population), andcollecting antisera from that immunized animal. A wide range of animalspecies can be used for the production of antisera. Typically the animalused for production of anti-antisera is a rabbit, a mouse, a rat, ahamster, a guinea pig or a goat. Because of the relatively large bloodvolume of rabbits, a rabbit is a preferred choice for production ofpolyclonal antibodies.

[0308] As is well known in the art, a given composition may vary in itsimmunogenicity. It is often necessary therefore to boost the host immunesystem, as may be achieved by coupling a peptide or polypeptideimmunogen to a carrier. Exemplary and preferred carriers are keyholelimpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albuminssuch as ovalbumin, mouse serum albumin or rabbit serum albumin can alsobe used as carriers. Means for conjugating a polypeptide to a carrierprotein are well known in the art and include glutaraldehyde,m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimyde andbis-biazotized benzidine.

[0309] As is also well known in the art, the immunogenicity of aparticular immunogen composition can be enhanced by the use ofnon-specific stimulators of the immune response, known as adjuvants.Exemplary and preferred adjuvants include complete Freund's adjuvant (anon-specific stimulator of the immune response containing killedMycobacterium tuberculosis), incomplete Freund's adjuvants and aluminumhydroxide adjuvant.

[0310] The amount of immunogen composition used in the production ofpolyclonal antibodies varies upon the nature of the immunogen as well asthe animal used for immunization. A variety of routes can be used toadminister the immunogen (subcutaneous, intramuscular, intradermal,intravenous and intraperitoneal). The production of polyclonalantibodies may be monitored by is sampling blood of the immunized animalat various points following immunization. A second, booster injection,may also be given. The process of boosting and titering is repeateduntil a suitable titer is achieved. When a desired titer level isobtained, the immunized animal can be bled and the serum isolated andstored, and/or the animal can be used to generate MAbs.

[0311] MAbs may be readily prepared through use of well-knowntechniques, such as those exemplified in *U.S. Pat. No. 4,196,265,incorporated herein by reference. Typically, this technique involvesimmunizing a suitable animal with a selected immunogen composition,e.g., a purified or partially purified tumor cell or vascularendothelial cell protein, polypeptide, peptide, or intact cellcomposition. The immunizing composition is administered in a mannereffective to stimulate antibody producing cells. Rodents such as miceand rats are preferred animals, however, the use of rabbit, sheep frogcells is also possible. The use of rats may provide certain advantages(*Goding, 1986, pp. 60-61), but mice are preferred, with the BALB/cmouse being most preferred as this is most routinely used and generallygives a higher percentage of stable fusions.

[0312] Following immunization, somatic cells with the potential forproducing antibodies, specifically B lymphocytes (B cells), are selectedfor use in the MAb generating protocol. These cells may be obtained frombiopsied spleens, tonsils or lymph nodes, or from a peripheral bloodsample. Spleen cells and peripheral blood cells are preferred, theformer because they are a rich source of antibody-producing cells thatare in the dividing plasmablast stage, and the latter because peripheralblood is easily accessible. Often, a panel of animals will have beenimmunized and the spleen of animal with the highest antibody titer willbe removed and the spleen lymphocytes obtained by homogenizing thespleen with a syringe. Typically, a spleen from an immunized mousecontains approximately 5×10⁷ to 2×10⁸ lymphocytes.

[0313] The antibody-producing B lymphocytes from the immunized animalare then fused with cells of an immortal myeloma cell, generally one ofthe same species as the animal that was immunized. Myeloma cell linessuited for use in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render then incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas).

[0314] Any one of a number of myeloma cells may be used, as are known tothose of skill in the art (*Goding, pp. 65-66, 1986; *Campbell, pp.75-83, 1984). For example, where the immunized animal is a mouse, onemay use P3-X63/Ag8, X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U,MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bul; for rats, one may useR210.RCY3, Y3-Ag 1.2.3, IR983F, 4B210 or one of the above listed mousecell lines; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6, are alluseful in connection with human cell fusions.

[0315] One preferred murine myeloma cell is the A63-A68, 653 myelomacell line, which is readily available from the ATCC. Another mousemyeloma cell line that may be used is the 8-azaguanine-resistant mousemurine myeloma SP2/0 non-producer cell line.

[0316] Methods for generating hybrids of antibody-producing spleen orlymph node cells and myeloma cells usually comprise mixing somatic cellswith myeloma cells in a 4:1 proportion, though the proportion may varyfrom about 20:1 to about 1:1, respectively, in the presence of an agentor agents (chemical or electrical) that promote the fusion of cellmembranes. Fusion methods using Sendai virus have been described byKohler & Milstein (1975; 1976), and those using polyethylene glycol(PEG), such as 37% (v/v) PEG, by Gefter et al. (1977). The use ofelectrically induced fusion methods is also appropriate (*Goding pp.71-74, 1986).

[0317] Fusion procedures usually produce viable hybrids at lowfrequencies, about 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose aproblem, as the viable, fused hybrids are differentiated from theparental, unfused cells (particularly the unfused myeloma cells thatwould normally continue to divide indefinitely) by culturing in aselective medium. The selective medium is generally one that contains anagent that blocks the de novo synthesis of nucleotides in the tissueculture media. Exemplary and preferred agents are aminopterin,methotrexate, and azaserine. Aminopterin and methotrexate block de novosynthesis of both purines and pyrimidines, whereas azaserine blocks onlypurine synthesis. Where aminopterin or methotrexate is used, the mediais supplemented with hypoxanthine and thymidine as a source ofnucleotides (HAT medium). Where azaserine is used, the media issupplemented with hypoxanthine.

[0318] The preferred selection medium is HAT. Only cells capable ofoperating nucleotide salvage pathways are able to survive in HAT medium.The myeloma cells are defective in key enzymes of the salvage pathway,e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannotsurvive. The B cells can operate this pathway, but they have a limitedlife span in culture and generally die within about two weeks.Therefore, the only cells that can survive in the selective media arethose hybrids formed from myeloma and B cells.

[0319] This culturing provides a population of hybridomas from whichspecific hybridomas are selected. Typically, selection of hybridomas isperformed by culturing the cells by single-clone dilution in microtiterplates, followed by testing the individual clonal supernatants (afterabout two to three weeks) for the desired reactivity. The assay shouldbe sensitive, simple and rapid, such as radioimmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays, dot immunobindingassays, and the like.

[0320] The selected hybridomas would then be serially diluted and clonedinto individual antibody-producing cell lines, which clones can then bepropagated indefinitely to provide MAbs. The cell lines may be exploitedfor MAb production in two basic ways. A sample of the hybridoma can beinjected (often into the peritoneal cavity) into a histocompatibleanimal of the type that was used to provide the somatic and myelomacells for the original fusion. The injected animal develops tumorssecreting the specific monoclonal antibody produced by the fused cellhybrid. The body fluids of the animal, such as serum or ascites fluid,can then be tapped to provide MAbs in high concentration. The individualcell lines could also be cultured in vitro, where the MAbs are naturallysecreted into the culture medium from which they can be readily obtainedin high concentrations. MAbs produced by either means may be furtherpurified, if desired, using filtration, centrifugation and variouschromatographic methods such as HPLC or affinity chromatography.

[0321] The inventors also contemplate the use of a molecular cloningapproach to generate monoclonals. For this, combinatorial immunoglobulinphagemid libraries are prepared from RNA isolated from the spleen of theimmunized animal, and phagemids expressing appropriate antibodies areselected by panning using cells expressing the antigen and control cellse.g., normal-versus-tumor cells. The advantages of this approach overconventional hybridoma techniques are that approximately 10⁴ times asmany antibodies can be produced and screened in a single round, and thatnew specificities are generated by H and L chain combination whichfurther increases the chance of finding appropriate antibodies.

[0322] Where MAbs are employed in the present invention, they may be ofhuman, murine, monkey, rat, hamster, chicken or even rabbit origin. Theinvention contemplates the use of human antibodies, “humanized” orchimeric antibodies from mouse, rat, or other species, bearing humanconstant and/or variable region domains, and other recombinantantibodies and fragments thereof. Of course, due to the ease ofpreparation and ready availability of reagents, murine monoclonalantibodies will typically be preferred.

[0323] 2. Functional Antibody Binding Regions

[0324] The origin or derivation of the targeting agent antibody orantibody fragment (e.g., Fab′, Fab, F(ab′)₂, Fv or scFv) is not believedto be particularly crucial to the practice of the invention, so long asthe antibody or fragment that is actually employed for the preparationof the bispecific ligand exhibits the desired binding properties.

[0325] When assaying antigens that may contain Fc receptors, such asmembrane antigens of lymphoid or reticuloendothelial origin, the use ofwhole antibody labels may result in unacceptably high levels of‘non-specific binding’. It may then be necessary to use antibodypreparations in which the Fc portion has been removed. Fragmentation ofimmunoglobulin molecules can be achieved by controlled proteolysis,although the conditions will vary considerably with species andimmunoglobulin class or subclass. Bivalent F(ab′)₂ fragments are usuallypreferable over the univalent Fab or Fab′ fragments.

[0326] Fab

[0327] Fab fragments can be obtained by proteolysis of the wholeimmunoglobulin by the non-specific thiol protease, papain. Papain mustfirst be activated by reducing the sulphydryl group in the active sitewith cysteine, 2-mercaptoethanol or dithiothreitol. Heavy metals in thestock enzyme should be removed by chelation with EDTA (2 mM) to ensuremaximum enzyme activity. Enzyme and substrate are normally mixedtogether in the ratio of 1:100 by weight. After incubation, the reactioncan be stopped by irreversible alkylation of the thiol group withiodoacetamide or simply by dialysis. The completeness of the digestionshould be monitored by SDS-PAGE and the various fractions separated byprotein A-Sepharose or ion exchange chromatography.

[0328] F(ab′)₂

[0329] The usual procedure for preparation of F(ab′)₂ fragments from IgGof rabbit and human origin is limited proteolysis by the enzyme pepsin(Protocol 7.3.2). The conditions, 100× antibody excess w/w in acetatebuffer at pH 4.5, 37° C., suggest that antibody is cleaved at theC-terminal side of the inter-heavy-chain disulfide bond. Rates ofdigestion of mouse IgG may vary with subclass and it may be difficult toobtain high yields of active F(ab′)₂ fragments without some undigestedor completely degraded IgG. In particular, IgG_(2b) is highlysusceptible to complete degradation. The other subclasses requiredifferent incubation conditions to produce optimal results.

[0330] Digestion of rat IgG by pepsin requires conditions includingdialysis in 0.1 M acetate buffer, pH 4.5, and then incubation for fourhours with 1% w/w pepsin; IgG₁ and IgG_(2a) digestion is improved iffirst dialysed against 0.1 M formate buffer, pH 2.8, at 4° C., for 16hours followed by acetate buffer. IgG_(2b) gives more consistent resultswith incubation in staphylococcal V8 protease (3% w/w) in 0.1 M sodiumphosphate buffer, pH 7.8, for four hours at 37° C.

[0331] 3. Bispecific Antibodies

[0332] In general, the preparation of bispecific antibodies is also wellknown in the art, as exemplified by Glennie et al. (1987). Bispecificantibodies have been employed clinically, for example, to treat cancerpatients (Bauer et al., 1991). One method for the preparation ofbispecific antibodies involves the separate preparation of antibodieshaving specificity for the targeted tumor cell antigen, on the one hand,and the coagulating agent (or other desired target, such as anactivating antigen) on the other.

[0333] Bispecific antibodies have also been developed particularly foruse as immunotherapeutic agents. As mentioned earlier in conjunctionwith antigen-induction, certain of these antibodies were developed tocross-link lymphocytes and tumor antigens (Nelson, 1991; Segal et al.,1992). Examples include chimeric molecules that bind T cells, e.g., atCD3, and tumor antigens, and trigger lymphocyte-activation by physicallycross-linking the TCR/CD3, complex in close proximity to the target cell(Staerz et al., 1985; Perez et al., 1985; 1986a; 1986b; Ting et al.,1988).

[0334] Indeed, tumor cells of carcinomas, lymphomas, leukemias andmelanomas have been reported to be susceptible to bispecificantibody-mediated killing by T cells (Nelson, 1991; Segal et al., 1992;deLeij et al., 1991). These type of bispecific antibodies have also beenused in several Phase I clinical trials against diverse tumor targets.Although they are not novel compositions in accordance with thisinvention, the combined use of bispecific cross-linking antibodies alongwith the bispecific coagulating ligands described herein is alsocontemplated. The bispecific cross-linking antibodies may beadministered as described in references such as deLeij et al. (1991);Clark et al. (1991); Rivoltini et al. (1992); Bolhuis et al. (1992); andNitta et al. (1990).

[0335] While numerous methods are known in the art for the preparationof bispecific antibodies, the Glennie et al. (1987) method involves thepreparation of peptic F(ab′γ)₂ fragments from the two chosen antibodies,followed by reduction of each to provide separate Fab′γ_(SH) fragments.The SH groups on one of the two partners to be coupled are thenalkylated with a cross-linking reagent such as o-phenylenedimaleimide toprovide free maleimide groups on one partner. This partner may then beconjugated to the other by means of a thioether linkage, to give thedesired F(ab′γ)₂ heteroconjugate.

[0336] Due to ease of preparation, high yield and reproducibility, theGlennie et al. (1987) method is often preferred for the preparation ofbispecific antibodies, however, there are numerous other approaches thatcan be employed and that are envisioned by the inventors. For example,other techniques are known wherein crosslinking with SPDP or protein Ais carried out, or a trispecific construct is prepared (Titus et al.,1987; Tutt et al., 1991).

[0337] Another method for producing bispecific antibodies is by thefusion of two hybridomas to form a quadroma (Flavell et al., 1991, 1992;Pimm et al., 1992; French et al., 1991; Embleton et al., 1991). As usedherein, the term “quadroma” is used to describe the productive fusion oftwo B cell hybridomas. Using now standard techniques, two antibodyproducing hybridomas are fused to give daughter cells, and those cellsthat have maintained the expression of both sets of clonotypeimmunoglobulin genes are then selected.

[0338] A preferred method of generating a quadroma involves theselection of an enzyme deficient mutant of at least one of the parentalhybridomas. This first mutant hybridoma cell line is then fused to cellsof a second hybridoma that had been lethally exposed, e.g., toiodoacetamide, precluding its continued survival. Cell fusion allows forthe rescue of the first hybridoma by acquiring the gene for its enzymedeficiency from the lethally treated hybridoma, and the rescue of thesecond hybridoma through fusion to the first hybridoma. Preferred, butnot required, is the fusion of immunoglobulins of the same isotype, butof a different subclass. A mixed subclass antibody permits the use if analternative assay for the isolation of a preferred quadroma.

[0339] In more detail, one method of quadroma development and screeninginvolves obtaining a hybridoma line that secretes the first chosen MAband making this deficient for the essential metabolic enzyme,hypoxanthine-guanine phosphoribosyltransferase (HGPRT). To obtaindeficient mutants of the hybridoma, cells are grown in the presence ofincreasing concentrations of 8-azaguanine (1×10⁻⁷M to 1×10⁻⁵M). Themutants are subcloned by limiting dilution and tested for theirhypoxanthine/aminopterin/thymidine (HAT) sensitivity. The culture mediummay consist of, for example, DMEM supplemented with 10% FCS, 2 mML-Glutamine and 1 mM penicillin-streptomycin.

[0340] A complementary hybridoma cell line that produces the seconddesired MAb is used to generate the quadromas by standard cell fusiontechniques (Galfre et al., 1981), or by using the protocol described byClark et al. (1988). Briefly, 4.5×10⁷ HAT-sensitive first cells aremixed with 2.8×10⁷ HAT-resistant second cells that have been pre-treatedwith a lethal dose of the irreversible biochemical inhibitoriodoacetamide (5 mM in phosphate buffered saline) for 30 minutes on icebefore fusion. Cell fusion is induced using polyethylene glycol (PEG)and the cells are plated out in 96 well microculture plates. Quadromasare selected using HAT-containing medium. Bispecific antibody-containingcultures are identified using, for example, a solid phaseisotype-specific ELISA and isotype-specific immunofluorescence staining.

[0341] In one identification embodiment to identify the bispecificantibody, the wells of microtiter plates (Falcon, Becton DickinsonLabware) are coated with a reagent that specifically interacts with oneof the parent hybridoma antibodies and that lacks cross-reactivity withboth antibodies. The plates are washed, blocked, and the supernatants(SNs) to be tested are added to each well. Plates are incubated at roomtemperature for 2 hours, the supernatants discarded, the plates washed,and diluted alkaline phosphatase-anti-antibody conjugate added for 2hours at room temperature. The plates are washed and a phosphatasesubstrate, e.g., P-Nitrophenyl phosphate (Sigma, St. Louis) is added toeach well. Plates are incubated, 3N NaOH is added to each well to stopthe reaction, and the OD₄₁₀ values determined using an ELISA reader.

[0342] In another identification embodiment, microtiter platespre-treated with poly-L-lysine are used to bind one of the target cellsto each well, the cells are then fixed, e.g. using 1% glutaraldehyde,and the bispecific antibodies are tested for their ability to bind tothe intact cell. In addition, FACS, immunofluorescence staining,idiotype specific antibodies, antigen binding competition assays, andother methods common in the art of antibody characterization may be usedin conjunction with the present invention to identify preferredquadromas.

[0343] Following the isolation of the quadroma, the bispecificantibodies are purified away from other cell products. This may beaccomplished by a variety of protein isolation procedures, known tothose skilled in the art of immunoglobulin purification. Means forpreparing and characterizing antibodies are well known in the art (See,e.g., *Antibodies: A Laboratory Manual, 1988).

[0344] For example, supernatants from selected quadromas are passed overprotein A or protein G sepharose columns to bind IgG (depending on theisotype). The bound antibodies are then eluted with, e.g. a pH 5.0citrate buffer. The elute fractions containing the BsAbs, are dialyzedagainst an isotonic buffer. Alternatively, the eluate is also passedover an anti-immunoglobulin-sepharose column. The BsAb is then elutedwith 3.5 M magnesium chloride. BsAbs purified in this way are thentested for binding activity by, e.g., an isotype-specific ELISA andimmunofluorescence staining assay of the target cells, as describedabove.

[0345] Purified BsAbs and parental antibodies may also be characterizedand isolated by SDS-PAGE electrophoresis, followed by staining withsilver or Coomassie. This is possible when one of the parentalantibodies has a higher molecular weight than the other, wherein theband of the BsAbs migrates midway between that of the two parentalantibodies. Reduction of the samples verifies the presence of heavychains with two different apparent molecular weights.

[0346] Furthermore, recombinant technology is now available for thepreparation of antibodies in general, allowing the preparation ofrecombinant antibody genes encoding an antibody having the desired dualspecificity (Van Duk et al., 1989). Thus, after selecting the monoclonalantibodies having the most preferred binding characteristics, therespective genes for these antibodies can be isolated, e.g., byimmunological screening of a phage expression library (Oi & Morrison,1986; Winter & Milstein, 1991). Then, through rearrangement of Fabcoding domains, the appropriate chimeric construct can be readilyobtained.

[0347] E. Binding Assays

[0348] Although the present invention has significant utility in animaland human treatment regimens, it also has many other practical uses.These uses are generally related to the specific binding ability of thebispecific compounds. In that all the compounds of the invention includeat least one targeting and binding component, e.g., an antibody, ligand,receptor, or such like, the resultant bispecific construct may be usedin virtually all of the binding embodiments that the original antibody,ligand or receptor, etc., may be used. The presence of the coagulant, orother binding regions, does not negate the utility of the first bindingregions in any binding assay.

[0349] As such, the bispecific coagulating ligands may be employed instandard binding assays, such as in immunoblots, Western blots, andother assays in which an antigen is immobilized onto a solid supportmatrix, e.g., nitrocellulose, nylon or a combination thereof. They maybe employed simply as an “antibody substitute” or may be used to providea more-specific detection means for use in detecting antigens againstwhich standard secondary reagents cause an unacceptably high background.This is especially useful when the antigens studied are themselvesimmunoglobulins or other antibodies are used in the procedure, asexemplified below in the case of ELISAs.

[0350] The bispecific binding ligands may also be used in conjunctionwith both fresh-frozen and formalin-fixed, paraffin-embedded tissueblocks in immunohistochemistry; in fluorescent activated cell sorting,flow cytometry or flow microfluorometry; in immunoprecipitation toseparate a target antigen from a complex mixture, in which case, due totheir potential to form molecular lattices, they may even achieveprecipitation without a secondary matrix-coupled reagent; in antigen orcell purification embodiments, such as affinity chromatography, evenincluding, in certain cases, the one-step rapid purification of one ormore cell populations at the same time; and in many other binding assaysthat will be known to those of skill in the art given the informationpresented herein.

[0351] As an example, the bispecific ligands of the invention may beused in ELISA assays. Many types of ELISAs are known and routinelypracticed in the art. The bispecific ligands may be employed in any ofthe binding steps, depending on the particular type of ELISA beingperformed and the “antigen” (component) to be detected. The ligandscould therefore be used to coat the plate, to compete for binding sites,as an antigen to provide a standard curve, as a primary binding ligand,as a secondary binding ligand or even as a tertiary or other bindingligand. The many modes of conducting ELISAs will be known to those ofskill in the art, in further light of the exemplary mode discussedbelow.

[0352] In one form of an ELISA, binding targets, generally antibodiesthemselves, are immobilized onto a selected surface, preferably asurface exhibiting a protein affinity such as the wells of a polystyrenemicrotiter plate. After washing to remove incompletely adsorbedmaterial, it is desirable to bind or coat the assay plate wells with anonspecific protein that is known to be antigenically neutral withregard to the test antisera such as bovine serum albumin (BSA), caseinor solutions of milk powder. This allows for blocking of nonspecificadsorption sites on the immobilizing surface and thus reduces thebackground caused by nonspecific binding of antisera onto the surface.

[0353] In these types of ELISAs, generally termed sandwich ELISAs, theplate-bound antibody is used to “trap” the antigen. After binding of thefirst antibody to the well, coating with a non-reactive material toreduce background, and washing to remove unbound material, theimmobilizing surface is contacted with, in the present exemplaryembodiment, a test sample containing the antigenic material to bedetected and/or titered in a manner conducive to immune complex(antigen/antibody).formation. These embodiments are particularly usefulfor detecting ligands in clinical samples or biological extracts. Thesamples are preferably diluted with solutions of BSA, bovine gammaglobulin (BGG) and phosphate buffered saline (PBS) and a detergent, e.g.Tween.

[0354] The layered antisera is then allowed to incubate for from 2 to 4hours, at temperatures preferably on the order of 25° to 37° C.Following incubation, the antisera-contacted surface is washed so as toremove non-immunocomplexed material. A preferred washing procedureincludes washing with a solution such as PBS/Tween, or borate buffer.

[0355] Following formation of specific immunocomplexes between the boundantigen and the test sample, and subsequent washing, the occurrence andamount of immunocomplex formation may be determined by subjecting samecomplex to a secondary specific binding component, which is generally anantibody-based component. In a particular embodiment, the bispecificligands of the invention are proposed for use in this step. Furtherspecific binding and washing steps are then conducted.

[0356] To provide a detecting means, in the present exemplaryembodiment, a third antibody is used that is linked to a detectablelabel, such as an associated enzyme that will generate a colordevelopment upon incubating with an appropriate chromogenic substrate.The third, or tertiary, labeled antibody has binding affinity for acomponent of the bispecific ligand. The ultimate immunocomplex isdetermined, after appropriate binding and washing steps, by detectingthe label, e.g., by incubating with a chromogenic substrate, such asurea and bromocresol purple or2,2′-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid [ABTS] and H₂O₂.Quantification is then achieved by measuring the degree of colorgeneration, e.g., using a visible spectra spectrophotometer.

[0357] Using a bispecific coagulating ligand as a secondary detectionreagent in conjunction with the type of ELISA described above hasdistinct advantages. For example, it allows the use of a tertiary,labeled antibody that is specific for a portion of the bispecific ligandthat is distinct from the typical antibody constant regions usuallytargeted. In particular, a tertiary binding ligand that is specific forthe coagulant portion (or coagulant binding region) of the bispecificconstruct may be employed. This novel means of detecting immune complexformation imparts improved specificity, which is particularly useful insandwich ELISAs where the tertiary antibody may cross-react with, andbind to, the original material used to coat the plate, i.e., theoriginal antibody, rather than just binding to the intended secondaryantibody. By directing the labelled tertiary component to annon-antibody portion, or even to a novel antigen combining region, of abispecific ligand, the problem of non-specific binding, and unusuallyhigh background, will be avoided.

[0358] Further practical uses of the bispecific ligands are evident byexploiting their coagulating ability. As all of the proposed compoundsare capable of inducing coagulation, they may be employed, e.g., as acontrol, in any assay that involves coagulation as a component. Thepresence of the targeting component does not negate the utility of thecoagulant in such assays, as each component functions independently ofthe other.

[0359] F. Effective Use of Tissue Factor-Binding Bispecific Antibodies

[0360] As mentioned earlier, tissue factor (TF) is one agent capable ofinitiating blood coagulation. TF is exposed to the blood in vasculardamage or following activation by certain cytokines. Available TF thencomplexes with factor VIIa to initiate the coagulation cascade thatultimately results in fibrin formation.

[0361] In one exemplary embodiment, the inventors have synthesized abispecific antibody with specificity for antigens on tumor vasculatureendothelial cells on one antigen combining site and specificity for theextracellular domains of human TF on the other antigen combining site.The antibody with specificity for human TF was previously shown to bindTF with high affinity without interfering with the factor VIIacomplexing event or the TF/VIIa activity (Morrissey et al., 1988).Instead of using full length human TF, the inventors used a truncatedform (tTF), which is devoid of the cytoplasmic as well as thetransmembrane domain. Truncated TF lacks coagulation inducing activity,while still being able to complex factor VIIa, probably because it isnot able to complex with a membrane surface upon which thecoagulation-initiation complexes, including Factor X, could assemble.

[0362] The mouse model used for analyzing the effectiveness of thistumor vasculature endothelial cell specific targeting construct was arecently established model in which MHC class II antigens, that areabsent from the vasculature of normal tissues, are expressed on thetumor vasculature through induction by IFN-γ that is secreted by thetumor cells (Burrows et al., 1992; Burrows & Thorpe, 1993). It has beendemonstrated that anti-class II antibody administered intravenouslylocalizes rapidly and strongly to the tumor vasculature (Burrows et al.,1992).

[0363] The present inventors herein demonstrate that, in a C1300 (Muγ)tumor bearing mouse, the anti-MHC Class II/anti-TF bispecific antibodyis able to induce coagulation specifically in the vasculature of thetumor when administered together with tTF. Indeed, intravenousadministration of the antibody:tTF complex induced rapid thrombosis oftumor vasculature and complete tumor regressions in 70% of animals.Neither the bispecific antibody alone, nor tTF alone, nor any of theisotype matched control antibodies in the presence or absence of tTF,was able to elicit the same effect. This indicates that the B21-2/10H10bispecific antibody acts as a “coaguligand” that is capable of bridgingtarget cells and tTF so that tTF can activate factor X and start thecoagulation cascade. It also shows the evident success of thecoaguligand in treating solid tumors.

[0364] G. Pharmaceutical Compositions and Kits

[0365] Pharmaceutical compositions of the present invention willgenerally comprise an effective amount of the bispecific coagulatingligand dissolved or dispersed in a pharmaceutically acceptable carrieror aqueous medium.

[0366] The phrases “pharmaceutically or pharmacologically acceptable”refer to molecular entities and compositions that do not produce anadverse, allergic or other untoward reaction when administered to ananimal, or a human, as appropriate. As used herein, “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents and the like. The use of such media and agents forpharmaceutical active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions.

[0367] 1. Parenteral Formulations

[0368] The bispecific ligands of the present invention will often beformulated for parenteral administration, e.g., formulated for injectionvia the intravenous, intramuscular, sub-cutaneous or other such routes,including direct instillation into a tumor or disease site. Thepreparation of an aqueous composition that contains a tumor-targetedcoagulant agent as an active ingredient will be known to those of skillin the art in light of the present disclosure. Typically, suchcompositions can be prepared as injectables, either as liquid solutionsor suspensions; solid forms suitable for using to prepare solutions orsuspensions upon the addition of a liquid prior to injection can also beprepared; and the preparations can also be emulsified.

[0369] Solutions of the active compounds as free base orpharmacologically acceptable salts can be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersions canalso be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

[0370] The pharmaceutical forms suitable for injectable use includesterile aqueous solutions or dispersions; formulations including sesameoil, peanut oil or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi.

[0371] The bispecific ligands or antibodies can be formulated into acomposition in a neutral or salt form. Pharmaceutically acceptablesalts, include the acid addition salts (formed with the free aminogroups of the protein) and which are formed with inorganic acids suchas, for example, hydrochloric or phosphoric acids, or such organic acidsas acetic, oxalic, tartaric, mandelic, and the like. Salts formed withthe free carboxyl groups can also be derived from inorganic bases suchas, for example, sodium, potassium, ammonium, calcium, or ferrichydroxides, and such organic bases as isopropylamine, trimethylamine,histidine, procaine and the like.

[0372] The carrier can also be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and vegetable oils. The proper fluidity canbe maintained, for example, by the use of a coating, such as lecithin,by the maintenance of the required particle size in the case ofdispersion and by the use of surfactants. The prevention of the actionof microorganisms can be brought about by various antibacterial adantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

[0373] Sterile injectable solutions are prepared by incorporating theactive compounds in the required amount in the appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby filtered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus nyadditional desired ingredient from a previously sterile-filteredsolution thereof.

[0374] Upon formulation, solutions will be administered in a mannercompatible with the dosage formulation and in such amount as istherapeutically effective. Formulations are easily administered in avariety of dosage forms, such as the type of injectable solutionsdescribed above, but drug release capsules and the like can also beemployed.

[0375] Suitable pharmaceutical compositions in accordance with theinvention will generally include an amount of the bispecific ligandadmixed with an acceptable pharmaceutical diluent or excipient, such asa sterile aqueous solution, to give a range of final concentrations,depending on the intended use. The techniques of preparation aregenerally well known in the art as exemplified by Remington'sPharmaceutical Sciences, 16th Ed. Mack Publishing Company, 1980,incorporated herein by reference. It should be appreciated thatendotoxin contamination should be kept minimally at a safe level, forexample, less that 0.5 ng/mg protein. Moreover, for humanadministration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiological Standards.

[0376] The therapeutically effective doses are readily determinableusing an animal model, as shown in the studies detailed herein (see,e.g., Example III). Experimental animals bearing solid tumors arefrequently used to optimize appropriate therapeutic doses prior totranslating to a clinical environment. Such models are known to be veryreliable in predicting effective anti-cancer strategies. For example,mice bearing solid tumors, such as used in Example III, are widely usedin pre-clinical testing.

[0377] The inventors have used mice with C1300 (Mo8) tumors to determinetoxicity limits and working ranges of bispecific that give optimalanti-tumor effects with minimal toxicity.

[0378] It is currently proposed that effective doses for use in thetreatment of cancer will be between about 0.1 mg/kg and about 2 mg/kg,and preferably, of between about 0.8 mg/kg and about 1.2 mg/kg, whenadministered via the IV route at a frequency of about 1 time per week.Some variation in dosage will necessarily occur depending on thecondition of the subject being treated. The person responsible foradministration will, in any event, determine the appropriate dose forthe individual subject. Such optimization and adjustment is routinelycarried out in the art and by no means reflects an undue amount ofexperimentation.

[0379] It should be remembered that one aspect of the present inventionconcerns the delivery of a coagulating agent to a tumor site byadministering an uncomplexed bispecific binding ligand that garners anendogenous coagulation factor from the circulation and concentrates itwithin the tumor site. In these cases, the pharmaceutical compositionsemployed will contain a ligand having a targeting and coagulant bindingregion, but will otherwise generally be the same as those describedabove.

[0380] In addition to the compounds formulated for parenteraladministration, such as intravenous or intramuscular injection, otherpharmaceutically acceptable forms are also contemplated, e.g., tabletsor other solids for oral administration, time release capsules,liposomal forms and the like. Other pharmaceutical formulations may alsobe used, dependent on the condition to be treated. For example, topicalformulations that are appropriate for treating pathological conditionssuch as dermatitis and psoriasis; and ophthalmic formulations fordiabetic retinopathy.

[0381] 2. Ingestible Formulations

[0382] In certain embodiments, active compounds may be administeredorally. This is contemplated for agents that are generally resistant, orhave been rendered resistant, to proteolysis by digestive enzymes. Suchcompounds are contemplated to include chemically designed or modifiedagents; dextrorotatory peptidyl agents; liposomal formulations; andformulations in time release capsules to avoid peptidase and lipasedegradation.

[0383] For oral administration, the active bispecific compounds may beadministered, for example, with an inert diluent or with an assimilableedible carrier, or they may be enclosed in hard or soft shell gelatincapsule, or compressed into tablets, or incorporated directly with thefood of the diet. For oral therapeutic administration, the activecompounds may be incorporated with excipients and used in the form ofingestible tablets, buccal tables, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. Such compositions andpreparations should contain at least 0.1% of active bispecificcoagulant. The percentage of the compositions and preparations may, ofcourse, be varied and may conveniently be between about 2 to about 60%of the weight of the unit. The amount of active compounds in suchtherapeutically useful compositions is such that a suitable dosage willbe obtained.

[0384] The tablets, troches, pills, capsules and the like may alsocontain the following: a binder, as gum tragacanth, acacia, cornstarch,or gelatin; excipients, such as dicalcium phosphate; a disintegratingagent, such as corn starch, potato starch, alginic acid and the like; alubricant, such as magnesium stearate; and a sweetening agent, such assucrose, lactose or saccharin may be added or a flavoring agent, such aspeppermint, oil of wintergreen, or cherry flavoring. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, a liquid carrier.

[0385] Various other materials may be present as coatings or tootherwise modify the physical form of the dosage unit. For instance,tablets, pills, or capsules may be coated with shellac, sugar or both. Asyrup of elixir may contain the active compounds sucrose as a sweeteningagent methyl and propylparabens as preservatives, a dye and flavoring,such as cherry or orange flavor. Of course, any material used inpreparing any dosage unit form should be pharmaceutically pure andsubstantially non-toxic in the amounts employed. In addition, the activecompounds may be incorporated into sustained-release preparation andformulations.

[0386] 3. Liposomal Formulations

[0387] The bispecific coagulating ligands of the present invention mayalso be formulated in liposomal preparations if desired. The followinginformation may be utilized in generating liposomal formulationsincorporating the present coagulants. Phospholipids form liposomes whendispersed in water, depending on the molar ratio of lipid to water. Thephysical characteristics of liposomes depend on pH, ionic strength andthe presence of divalent cations. Liposomes can show low permeability toionic and polar substances, but at elevated temperatures undergo a phasetransition which markedly alters their permeability. The phasetransition involves a change from a closely packed, ordered structure,known as the gel state, to a loosely packed, less-ordered structure,known as the fluid state. This occurs at a characteristicphase-transition temperature and results in an increase in permeabilityto ions, sugars and drugs.

[0388] In addition to temperature, exposure to proteins can alter thepermeability of liposomes. Certain soluble proteins such as cytochrome cbind, deform and penetrate the bilayer, thereby causing changes inpermeability. Cholesterol inhibits this penetration of proteins,apparently by packing the phospholipids more tightly. It is contemplatedthat the most useful liposome formations for use with the presentinvention will contain cholesterol, or even PEG.

[0389] The ability to trap solutes varies between different types ofliposomes. For example, multilamellar vesicles (MLVs) are moderatelyefficient at trapping solutes, but small unilamellar vesicles (SUVs) areinefficient. SUVs offer the advantage of homogeneity and reproducibilityin size distribution, however, and a compromise between size andtrapping efficiency is offered by large unilamellar vesicles (LUVs).These are prepared by ether evaporation and are three to four times moreefficient at solute entrapment than MLVs.

[0390] In addition to liposome characteristics, an important determinantin entrapping compounds is the physicochemical properties of thecompound itself. Polar compounds are trapped in the aqueous spaces andnonpolar compounds bind to the lipid bilayer of the vesicle. Polarcompounds are released through permeation or when the bilayer is broken,but nonpolar compounds remain affiliated with the bilayer unless it isdisrupted by temperature or exposure to lipoproteins. Both types showmaximum efflux rates at the phase transition temperature.

[0391] Liposomes interact with cells via four different mechanisms:Endocytosis by phagocytic cells of the reticuloendothelial system suchas macrophages and neutrophils; adsorption to the cell surface, eitherby nonspecific weak hydrophobic or electrostatic forces, or by specificinteractions with cell-surface components; fusion with the plasma cellmembrane by insertion of the lipid bilayer of the liposome into theplasma membrane, with simultaneous release of liposomal contents intothe cytoplasm; and by transfer of liposomal lipids to cellular orsubcellular membranes, or vice versa, without any association of theliposome contents. It often is difficult to determine which mechanism isoperative and more than one may operate at the same time.

[0392] The fate and disposition of intravenously injected liposomesdepend on their physical properties, such as size, fluidity and surfacecharge. They may persist in tissues for hours or days, depending ontheir composition, and half lives in the blood range from minutes toseveral hours. Larger liposomes, such as MLVs and LLTs, are taken uprapidly by phagocytic cells of the reticuloendothelial system, butphysiology of the circulatory system restrains the exit of such largespecies at most sites. They can exit only in places where large openingsor pores exist in the capillary endothelium, such as the sinusoids ofthe liver or spleen. Thus, these organs are the predominate site ofuptake. On the other hand, SUVs show a broader tissue distribution butstill are sequestered highly in the liver and spleen. In general, thisin vivo behavior dictates that liposomes concentrate only in thoseorgans and tissues accessible to their large size. As this clearlyincludes the blood, this is not a limitation to their combined use withthe present invention.

[0393] In other embodiments, the bispecific components of the inventionmay be admixed with the liposome surface to direct the drug contents tothe specific antigenic receptors located on the target cell surface.Carbohydrate determinants (glycoprotein or glycolipid cell-surfacecomponents that play a role in cell-cell recognition, interaction andadhesion) may also be used as recognition sites as they have potentialin directing liposomes to particular cell types. Mostly, it iscontemplated that intravenous injection of liposomal preparations wouldbe used, but other routes of administration are also conceivable.

[0394] 4. Topical Formulations

[0395] The formulation of bispecific coagulants for topical use, such asin creams, ointments and gels is also contemplated. The preparation ofoleaginous or water-soluble ointment bases is also well known to thosein the art. For example, these compositions may include vegetable oils,animal fats, and more preferably, semisolid hydrocarbons obtained frompetroleum. Particular components used may include white ointment, yellowointment, cetyl esters wax, oleic acid, olive oil, paraffin, petrolatum,white petrolatum, spermaceti, starch glycerite, white wax, yellow wax,lanolin, anhydrous lanolin and glyceryl monostearate.

[0396] Various water-soluble ointment bases may also be used, includingglycol ethers and derivatives, polyethylene glycols, polyoxyl 40stearate and polysorbates. Even delivery through the skin may beemployed if desired, e.g., by using transdermal patches, iontophoresisor electrotransport.

[0397] 5. Ophthalmic Formulations

[0398] The bispecific coagulating ligands of the present invention mayalso be formulated into pharmaceutical compositions suitable for use asophthalmic solutions. Such ophthalmic solutions are of interest, forexample, in the treatment of diabetic retinopathy. Thus, for thetreatment of diabetic retinopathy a bispecific conjugate of thisinvention would be administered to the eye of the subject in need oftreatment in the form of an ophthalmic preparation prepared inaccordance with conventional pharmaceutical practice, see for example“Remington's Pharmaceutical Sciences” 15th Edition, pages 1488 to 1501(Mack Publishing Co., Easton, Pa.).

[0399] The ophthalmic preparation will contain a novel bispecificcoagulant or a pharmaceutically acceptable salt thereof in aconcentration from about 0.01 to about 1% by weight, preferably fromabout 0.05 to about 0.5% in a pharmaceutically acceptable solution,suspension or ointment. Some variation in concentration will necessarilyoccur, depending on the particular compound employed, the condition ofthe subject to be treated and the like, and the person responsible fortreatment will determine the most suitable concentration for theindividual subject. The ophthalmic preparation will preferably be in theform of a sterile aqueous solution containing, if desired, additionalingredients, for example preservatives, buffers, tonicity agents,antioxidants and stabilizers, nonionic wetting or clarifying agents,viscosity-increasing agents and the like.

[0400] Suitable preservatives for use in such a solution includebenzalkonium chloride, benzethonium chloride, chlorobutanol, thimerosaland the like. Suitable buffers include boric acid, sodium and potassiumbicarbonate, sodium and potassium borates, sodium and potassiumcarbonate, sodium acetate, sodium biphosphate and the like, in amountssufficient to maintain the pH at between about pH 6 and pH 8, andpreferably, between about pH 7 and pH 7.5. Suitable tonicity agents aredextran 40, dextran 70, dextrose, glycerin, potassium chloride,propylene glycol, sodium chloride, and the like, such that the sodiumchloride equivalent of the ophthalmic solution is in the range 0.9 plusor minus 0.20%.

[0401] Suitable antioxidants and stabilizers include sodium bisulfite,sodium metabisulfite, sodium thiosulfite, thiourea and the like.Suitable wetting and clarifying agents include polysorbate 80,polysorbate 20, poloxamer 282 and tyloxapol. Suitableviscosity-increasing agents include dextran 40, dextran 70, gelatin,glycerin, hydroxyethylcellulose, hydroxmethylpropylcellulose, lanolin,methylcellulose, petrolatum, polyethylene glycol, polyvinyl alcohol,polyvinylpyrrolidone, carboxymethylcellulose and the like. Theophthalmic preparation will be administered topically to the eye of thesubject in need of treatment by conventional methods, for example in theform of drops or by bathing the eye in the ophthalmic solution.

[0402] 6. Therapeutic Kits

[0403] The present invention also provides therapeutic kits comprisingthe bispecific coagulating ligands described herein. Such kits willgenerally contain, in suitable container means, a pharmaceuticallyacceptable formulation of at least one bispecific ligand in accordancewith the invention. The kits may also contain other pharmaceuticallyacceptable formulations, such as those containing additional bispecificcoagulating ligands, generally those having a distinct targetingcomponent; extra uncomplexed coagulation factors; bispecific antibodies,T cells, or other functional components for use in, e.g., antigeninduction; components for use in antigen suppression, such as acyclosporin; distinct anti-tumor site antibodies or immuntoxins; and anyone or more of a range of chemotherapeutic drugs.

[0404] Preferred agents for use in combination kits are inducing agentscapable of inducing disease-associated vascular endothelial cells toexpress a targetable antigen, such as E-selectin or an MHC Class IIantigen. Inducing agents can include T cell clones that bind disease ortumor antigens and that produce IFN-γ. Preferred inducing agents includebispecific antibodies that bind to disease or tumor cell antigens and toeffector cells capable of inducing target antigen expression through theelaboration of cytokines.

[0405] As such, the present invention further includes kits thatcomprise, in suitable container means, a first pharmaceuticalcomposition comprising a bispecific antibody that binds to an activatingantigen on an effector cell surface, i.e., a monocyte/macrophage, mastcell, T cell or NK cell, and to an antigen on the cell surface ofdisease cell; and a second pharmaceutical composition comprising abispecific ligand that comprises a first binding region that binds to anendothelial cell antigen induced by an activated effector cell, orcytokine therefrom, where the first binding region is operatively linkedto a coagulation factor or a second binding region that binds to acoagulation factor.

[0406] Kits including a first pharmaceutical composition that comprisesa bispecific antibody that binds to the activating antigen CD14, CD16(FcR for IgE), CD2, CD3, CD28 or the T-cell receptor antigen arepreferred, with CD14 or CD28 binding bispecific antibodies being morepreferred. Activation of monocyte/macrophages or mast cells via CD14 orCD16 binding results in IL-1 production that induces E-selectin; whereasactivation of T cells via CD2, CD3 or CD28 binding results in IFN-γproduction that induces MHC class II. Kits that include a secondpharmaceutical composition that comprises a bispecific ligand thatcomprises a first binding region that binds to E-selectin or to an MHCClass II antigen are therefore also preferred.

[0407] The kits may have a single container means that contains thebispecific coagulating ligand, with or without any additionalcomponents, or they may have distinct container means for each desiredagent. Kits comprising the separate components necessary to make abispecific coagulating ligand are also contemplated.

[0408] When the components of the kit are provided in one or more liquidsolutions, the liquid solution is an aqueous solution, with a sterileaqueous solution being particularly preferred. However, the componentsof the kit may be provided as dried powder(s). When reagents orcomponents are provided as a dry powder, the powder can be reconstitutedby the addition of a suitable solvent. It is envisioned that the solventmay also be provided in another container means.

[0409] The container means of the kit will generally include at leastone vial, test tube, flask, bottle, syringe or other container means,into which the bispecific coagulating ligand, and any other desiredagent, may be placed and, preferably, suitably aliquoted. Whereadditional components are included, the kit will also generally containa second vial or other container into which these are placed, enablingthe administration of separated designed doses. The kits may alsocomprise a second/third container means for containing a sterile,pharmaceutically acceptable buffer or other diluent.

[0410] The kits may also contain a means by which to administer thebispecific ligand to an animal or patient, e.g., one or more needles orsyringes, or even an eye dropper, pipette, or other such like apparatus,from which the formulation may be injected into the animal or applied toa diseased area of the body. The kits of the present invention will alsotypically include a means for containing the vials, or such like, andother component, in close confinement for commercial sale, such as,e.g., injection or blow-molded plastic containers into which the desiredvials and other apparatus are placed and retained.

[0411] The following examples are included to demonstrate preferredembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples that followrepresent techniques discovered by the inventors to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments that are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

EXAMPLE I Synthesis of a Bispecific Coagulating Antibody

[0412] The present example describes the synthesis of a bispecificantibody capable of specifically directing a coagulant to a tumor site,i.e., a “coaguligand”.

[0413] A. Materials and Methods

[0414] 1. Reagents

[0415] Pepsin (A; EC 3.4.23.1), Ellmans reagent (ER;5,5′-dithio-bis(2-nitrobenzoic acid, DNTB), 2-mercaptoethanol (2-ME),sodium arsenite (NaAsO₂) and rabbit brain thromboplastin (acetonepowder) were obtained from Sigma Chemical Co., St. Louis Mo. SephadexG-25 and G-100 were obtained from Pharmacia LKB (Piscataway, N.J.).

[0416] 2. Human Truncated Tissue Factor (tTF)

[0417] Recombinant human truncated TF (tTF) was prepared by one of twodifferent methods.

[0418] Method I: Construction of the E. coli Expression Vector. The cDNAcoding for tTF (residues 1-218) was amplified by PCR using the primers5′-GAAGAAGGGATCCTGGTGCCTCGTGGTTCTGGCACTACAAATACT-3′ (5′-primer; SEQ IDNO: 28) and 5′-CTGGCCTCAAGCTTAACGGAATTCACCTTT-3′ (3′-primer; SEQ ID NO:29) which allowed the addition of the coding sequence for a thrombincleavage site upstream of the cDNA. The PCR products were cleaved usingBamHI and HindIII and ligated between the BamHI and HindIII sites of theexpression vector pTrcHisC (Invitrogen).

[0419] DH5α cells were transformed with the ligation mixture andrecombinant plasmids were isolated after selection in the presence ofampicillin. The E. coli strain BL21 was transformed with the recombinantplasmid pTrcHisC-tTF and the resultant transformants were used forprotein expression.

[0420] Method I: Expression, Refolding and Purification of tTF from E.coli. The poly(his)-tTF fusion protein was expressed using BL21 cellstransformed with pTrc-HisC-tTF. Inoculant cultures (10 ml in LB medium)were grown overnight shaking at 37° C.

[0421] Inoculant cultures were added to growth medium which were thengrown shaking at 37° C. When the optical density at 550 nm had reach ca.0.5, 10 ml of 100 mM isopropyl-β-D-thiogalactopyranoside was added.Shaking was continued at 37° C. for ca. 20 h (to stationary phase).

[0422] The cells were harvested by centrifugation (10,000×g, 20 min.)and the inclusion bodies were isolated as follows (quantities ofreagents are per gram of cell paste). The cell paste was suspended in 4ml of 10 mM Tris, pH 7.5, 150 mM NaCl, 1 mM MgCl₂, 0.17 mg/ml PMSF, 2mg/ml hen egg white lysozyme (Sigma). Benzonase (250 units, EM Science)was added the suspension was mixed gently at room temperature for 1.5 hthen centrifuged at 12,000 g for 15 min.

[0423] The pellet was resuspended in 10 mM Tris, pH 7.5, 1 mM EDTA, 3%NP40 (2 ml), sonicated for 1 min at 50% power and centrifuged at12,000×g for 20 min. The pellet was resuspended in water, sonicated for20-30 seconds at 50% power and centrifuged at 12,000×g for 20 min. Thewater wash was repeated and the final pellet, highly enriched for theinclusion bodies, was suspended in 6 M guanidinium chloride, 0.5 M NaCl,20 mM phosphate, 10 mM β-mercaptoethanol, pH 8.0 (9 ml per gram ofinclusion bodies) by gentle mixing at room temperature overnight.

[0424] The suspension was centrifuged at 12,000×g for 20 min and thesupernatant was loaded onto a nickel nitriloacetic acid (Ni-NTA, Qiagen)column. The column was washed successively with the same 6 M guanidiniumchloride buffer at pH 8 then pH 7, then the protein was eluted bydecreasing the pH to 4.

[0425] Ni-NTA column fractions containing the fusion protein werecombined and dithiothreitol was added to 50 mM. The solution was held atroom temperature overnight then diluted to a protein concentration ofca. 1 mg/ml in 6 M urea, 50 mM Tris, 0.02% sodium azide, pH 8.0 anddialyzed at 4° C. overnight against 10-20 volumes of the same buffer.The buffer was changed to 2 M urea, 50 mM Tris, 300 mM NaCl, 2.5 mMreduced glutathione, 0.5 mM oxidized glutathione, 0.02% sodium azide, pH8.0 (folding buffer). Dialysis was continued for 2 more days, the bufferwas replaced by fresh folding buffer and dialysis was continued for 2more days.

[0426] The solution was then dialyzed extensively against 20 mM TEA (pH7.5), removed from the dialysis bag, treated with human thrombin (ca. 1part per 500 parts recombinant protein w/w) overnight at roomtemperature, and loaded onto a HR-10/10 mono-Q anion exchange column.tTF protein was eluted using a 20 mM TEA buffer containing NACl in aconcentration increasing linearly from 0 to 150 mM over 30 minutes (flowrate 3 ml/min).

[0427] Method II: Preparation of tTF complimentary DNA (cDNA). RNA fromJ-82 cells (human bladder carcinoma) was used for the cloning of tTF.Total RNA was isolated using the GlassMax™ RNA microisolation reagent(Gibco BRL). The RNA was reverse transcribed to cDNA using the GeneAmpRNA PCR kit (Perkin Elmer). tTF cDNA was amplified using the same kitwith the following two primers: 5′ primer: 5′GTC ATG CCA TGG CCC TGG TGCCTC GTG CTT CTG GCA CTA CAA ATA CT (SEQ ID NO:2) 3′ primer: 5′TGA CAAGCT TAT TCT CTG AAT TCC CCC TTT CT

[0428] The underlined sequences codes for the N-terminus and C-terminusof tTF. The rest of the sequence in the 5′ primer is the restrictionsite for NcoI allowing the cloning of tTF into the expression vector andcodes for a cleavage site for thrombin. The sequence in the 3′ primer isthe HindIII site for cloning tTF into the expression vector. PCRamplification was performed as suggested by the manufacturer. Briefly,75 μM dNTP; 0.6 μM primer, 1.5 mM MgCl₂ were used and 30 cycles of 30″at 95° C., 30″ at 55° C. and 30” at 72° C. were performed.

[0429] Method II. Vector Constructs. E. coli expression vector H₆ pQE-60was used for expressing tTF (Lee et. al., 1994). The PCR amplified tTFcDNA was inserted between the NcoI and HindIII site. H₆ pQE-60 has abuilt-in (His)₆ encoding sequence such that the expressed protein hasthe sequence of (His)₆ at the N-terminus, which can be purified on theNi-NTA column.

[0430] Method II. tTF Purification. tTF containing H₆ pQE-60 DNA wastransformed to E. coli TG-1 cells. The cells were grown to OD₆₀₀=0.5 andIPTG was added to 30 μM to induce the tTF production. The cells wereharvested after shaking for 18 h at 30° C. The cell pellet was denaturedin 6 M Gu-HCl and the lysate was loaded onto a Ni-NTA column (Qiagen).The bound tTF was washed with 6 M urea and tTF was refolded with agradient of 6 M−1 M urea at room temperature for 16 h. The column waswashed with wash buffer (0.05 Na H₂ PO₄, 0.3 M NaCl, 10% glycerol) andtTF was eluted with 0.2 M Imidozole in wash buffer.

[0431] The eluted tTF was concentrated and loaded onto a G-75 column.tTF monomers were collected and treated with thrombin to remove the H₆peptide. This was done by adding 1 part of thrombin (Sigma) to 500 partsof tTF (w/w), and the cleavage was carried out at room temperature for18 h. Thrombin was removed from tTF by passage of the mixture through aBenzamidine Sepharose 6B thrombin affinity column (Pharmacia).

[0432] The tTF had identical ability to recombinant tTF from yeast orChinese hamster ovary cells to bind factor VIIa and to enhance thecatalytic activity of VIIa (Ruf et al., 1991). When analyzed bypolyacrylamide gel electrophoresis in sodium dodecyl sulfate, it ran asa single component having a molecular weight of approximately 24 kD.

[0433] 3. Monoclonal Antibodies

[0434] B21-2 (TIB-229) hybridoma and SFR8-B6 hybridoma (HB-152,hereafter referred to as SFR8) were obtained from the ATCC. Bothhybridomas secreted rat IgG2b antibodies, which were purified fromculture supernatant by protein G affinity chromatography. The B21-2antibody reacts with I-A^(d) antigen expressed on A20 cells as well ason the vasculature of the C1300 (Muγ) transfectant tumors grown inBALB/c/nu/nu mice. SFR8 antibody is directed against the HLA-Bw6 epitopeand serves as an isotype matched negative control for the B21-2antibody.

[0435] TF9/10H10 (referred to as 10H10), a mouse IgG1, is reactive withhuman TF without interference of TF/factor VIIa activity and wasproduced as described by Morrissey et al. (1988).

[0436] The cell line MRC OX7 (referred to as OX7) was obtained from Dr.A. F. Williams (MRC Cellular Immunology Unit, University of Oxford,Oxford, England). It secretes the OX7 antibody, a mouse IgG1 antibodythat recognizes the Thy 1.1 antigen on T lymphocytes. It was used as anisotype matched negative control for TF9/10H10.

[0437] All antibodies were purified from culture supernatant by proteinG affinity chromatography.

[0438] 4. Synthesis of Bispecific Antibodies

[0439] F(ab′)₂ fragments were obtained by digesting their respectiveIgGs with 2% (w/v) pepsin for 5-9 hrs at 37° C. and purification of thefragments by Sephadex G100 chromatography. Synthesis of the bispecificantibodies B21-2/10H10, SFR8/10H10 and B21-2/OX7 was carried outaccording to the method of Brennan et al. (1985) with minormodifications.

[0440] The bispecific antibodies B21-2/10H10, SFR8/10H10, OX7/10H10 andB21-2/OX7 were synthesized according to the method of Brennan et al.(1985) with minor modifications. In brief, F(ab′)₂ fragments wereobtained from the IgG antibodies by digestion with pepsin and werepurified to homogeneity by chromatography Sephadex G100. F(ab′)₂fragments were reduced for 16 h at 20° C. with 5 mM 2-mercaptoethanol in0.1 M sodium phosphate buffer, pH 6.8, containing 1 mM EDTA (PBSEbuffer) and 9 mM NaAsO₂. Ellman's reagent (ER) was added to give a finalconcentration of 25 mM and, after 3 h at 20° C., theEllman's-derivatized Fab′ fragments (Fab′-ER) were separated fromunreacted ER on columns of Sephadex G25 in PBSE.

[0441] To form the bispecific antibody, Fab′-ER derived from oneantibody was concentrated to approximately 2.5 mg/ml in anultrafiltration cell and was reduced with 10 mM 2-mercaptoethanol for 1h at 20° C. The resulting Fab′-SH was filtered through a column ofSephadex G25 in PBSE and was mixed with equal molar quantities ofFab′-ER prepared from the second antibody. The mixtures wereconcentrated by ultrafiltration to approximately 3 mg/ml and werestirred for 16 h at 20° C. The products of the reaction werefractionated on columns of Sephadex G100 and the fractions containingthe bispecific antibody (110 kDa) were concentrated to 1 mg/ml, and werestored at 4° C. in 0.02% sodium azide.

[0442] B. Results

[0443] 1. Analysis of Bispecific Antibodies

[0444] The molecular weight of the F(ab′)₂ fragments and bispecificpreparations were determined by SDS-Page electrophoresis with 4-15%gradient gels using the Pharmacia LKB-Phastsystem (Pharmacia LKB,Piscataway, N.J.). Bispecificity as well as the percentage ofheterodimer vs homodimer was determined by FACS analysis (Example II).

[0445] Analysis of the bispecific antibodies by SDS-Page electrophoresis(and by FACS, Example II) demonstrated that the B21-2/10H10 bispecificcontained less than 4% homodimer of either origin and <10% fragmentswith a molecular weight of 140 kD or 55 kD. Approximately 10% of thepreparation consisted of 140 kD fragments, probably being a F(ab′)₂construct with an extra light chain (of either origin) attached.

EXAMPLE II Coagulating Antibody Binding and Function In Vitro

[0446] The present example shows the bispecificity of the coagulatingantibody (coaguligand) and demonstrates that specific binding, cellulardelivery and coagulation is achieved in vitro using the coaguligand.

[0447] A. Materials and Methods

[0448] 1. Cells

[0449] The A20 cell line, which is an I-A^(d) positive BALB/c B-celllymphoma, was purchased from the American Type Culture Collection (ATCC;Rockville, Md.; TIB-208). A20 cells were grown in DMEM supplemented with10% (v/v) fetal calf serum (FCS), 0.2 mM L-glutamine, 200 units/mlpenicillin and 100 μg/ml streptomycin, 18 mM Hepes, 0.1 mM non-essentialamino acids mix and 1 mM sodium pyruvate (medium hereafter referred toas complete DMEM; all reagents obtained from Life Technologies,Gaitherburg, Md.). 2-ME is added to complete DMEM to a finalconcentration of 0.064 mM for A20 cells. Cultures were maintained at 37°C. in a humidified atmosphere of 90% air/10% CO₂.

[0450] J82, a human gall bladder carcinoma expressing TF, was obtainedfrom the ATCC (HTB-1). The cells grew adherently in complete DMEM.

[0451] The C1300 neuroblastoma cell line was established from aspontaneous tumor, which arose in an A/Jax mouse (Dunham & Stewart,1953). The C1300 (Muγ) 12 line, hereafter referred to as C1300 (Muγ) wasderived by transfection of C1300 neuroblastoma cells with the murineIFN-γ gene using the IFN-γ expression retrovirus pSVX (Muγ ΔAs)(Watanabe et al., 1989). The IFN-γ expression retrovirus was obtainedfrom Dr. Y. Watanabe (Department of Molecular Microbiology, KyotoUniversity, Japan).

[0452] C1300(Muγ)12 cells were maintained in Dulbecco's modified EagleMedium (DMEM) supplemented with 10% (v/v) fetal calf serum (FCS), 2.4 mML-glutamine, 200 units/ml penicillin, 100 μg/ml streptomycin, 100 μMnonessential amino acids, 1 μM sodium pyruvate, 18 μM HEPES and 1 mg/mlG418 (Geneticin; Sigma). Cultures were maintained at 37° C. in ahumidified atmosphere of 90% air/10% CO₂.

[0453] The Thy 1.1-expressing AKR-A mouse T lymphoma cell line wasobtained from Prof. Dr. I. MacLennan (Department of ExperimentalPathology, Birmingham University, Birmingham, England) and were grown incomplete DMEM.

[0454] 2. Indirect Immunofluorescence

[0455] A20 cells were resuspended in PBS/0.20% BSA/0.02% Na-azide(hereafter referred to as FACS buffer) at 4×10⁶ cells/ml. J82 cells werereleased from the flask under mild conditions using PBS/EDTA (0.2 % w/v)and resuspended at 4×10⁶ cells/ml in FACS buffer. 50 μl of cellsuspension was added to 50 μl of optimal serial dilutions of the primaryantibody in wells of a round-bottomed 96 well plate. After incubation atRT for 15 min, the cells were washed with FACS buffer 3 times. Afterremoving the final supernatant, 50 μl of the secondary antibodyconjugated to fluorescein isothiocyanate (FITC), in a 1 in 20 dilutionin FACS buffer, was added to the cells. The cells were incubated for afurther 15 min at RT and washed 3 times with FACS buffer. Cellassociated fluorescence was measured on a FACScan (Becton Dickenson,Fullerton, Calif.). Data were analyzed using the Lysis II program. WhenFITC-anti-rat immunoglobulin was used as the secondary antibody, normalmouse serum (10% v/v) was added to block non-specific cross reactivitywith the mouse cells.

[0456] 3. Radiolabeling of Proteins

[0457] Proteins were labeled with ¹²⁵Iodine according to the chloramineT protocol described by Mason & Williams (1980), (protocol 2). Theiodinated product was purified on G25 and stored at −70° C. in thepresence of 5% DMSO and 5 mg/ml bovine IgG in the case of the monoclonalfragments and 5% DMSO and 5 mg/ml BSA in the case of tTF. Specificactivity ranged between 2.5 μCi/g and 4.8 μCi/μg.

[0458] 4. Binding Studies

[0459] Human tTF was labelled with ¹²⁵I to a specific activity of2.5-4.8 μCi/μg using the chloramine T procedure (Protocol 2) describedby Mason and Williams (1980). A suspension of A20 cells at 2×10⁶cells/ml in PBS containing 2 mg/ml BSA and 0.02% sodium azide wasdistributed in 50 μl volumes into the wells of 96 well round-bottomedmicrotiter plates. To the wells were added 25 μl of bispecificantibodies prepared over a range of concentrations (8 to 0.02 μg/ml) inthe same buffer.

[0460] 25 μl of ¹²⁵I-tTF at 8 μg/ml in the same buffer were added toeach well, giving a molar excess of tTF. The plates were shaken andincubated for 1 hr at 4° C. The cells were then washed 3× in the plateswith 0.9% (w/v) NaCl containing 2 mg/ml BSA. The contents of the wellswere pipetted over a 10:11 (v/v) mixture of dibutyl phthalate andbis(2-ethylhexyl)phthalate oils in microcentrifuge tubes. The tubes werecentrifuged for 1.5 min at 7500 g and were snap frozen in liquidnitrogen. The tips containing the cells were cut off. The radioactivityin the cell pellet and in the supernatant was measured in a gammacounter.

[0461] 5. Coaculation Assay

[0462] An identical microplate to that used for the binding assay abovewas set up on the same occasion, except that non-labelled tTF was addedinstead of ¹²⁵I-tTF. After the 1 h incubation at 4° C., the cells werewashed 3× as before and were resuspended in 75 μl of 0.9% NaClcontaining 2 mg/ml BSA and 12.5 mM CaCl₂. The contents of the wells weretransferred to 5 ml clear plastic tubes and were warmed to 37° C. Toeach tube was added 30 μl of citrated mouse plasma at 37° C. The timefor the first fibrin strands to form was recorded.

[0463] B. Results

[0464] 1. Antibody Bispecificity

[0465] For SFR8/10H10 bispecificity was shown by FACS using J82 cells(TF positive) as target cells and FITC-anti-mouse immunoglobulin todemonstrate 10H10 presence. FITC-anti-rat immunoglobulin was used todemonstrate the presence of SFR8. The mean fluorescenceintensity-versus-concentration curves were coincident for both stains,demonstrating that both the mouse and the rat arm are present in thebispecific preparation.

[0466] 2. Antibody Binding

[0467] Binding studies with ¹²⁵Iodine labeled B21-2 Fab′ and SFR8 Fab′showed that the concentration at which saturation of binding of B21-2Fab′ to A20 cells is reached is 21.5 nM. The SFR8 Fab′ boundnon-specifically to A20 cells, with the number of molecules bound percell being less than 50,000 at 21.5 nM versus 530,000 for B21-2 Fab′.

[0468] 3. Coaculant Delivery and Tethering

[0469] To study the capability of bridging tTF to A20 cells through theB21-2/10H10 bispecific antibody as compared to the control bispecificantibodies, A20 cells were incubated with bispecific antibody and a¹²⁵I-tTF concentration range as indicated. Saturation was attained atconcentrations of bispecific antibody of 10 nM (1 μg/ml) or more, whenan average of 310,000 molecules of tTF were bound to each A20 cell. Thebinding was specific since no tTF binding was mediated by either of theisotype-matched control bispecific antibodies, SFR8/10H10 or B21-2/OX7,which had only one of the two specificities needed for tethering tTF(FIG. 1).

[0470] 4. Coaculation

[0471] To investigate whether tTF bound to A20 cells through abispecific antibody was able to induce coagulation, the inventors firstincubated A20 cells with 21.5 nM bispecific antibody and 69 nM tTF. Theresulting effect on the coagulation time is shown in Table VII. Thesefirst studies showed that A20 cells coated with a complex of B21-2/10H10and tTF were capable of inducing fibrin formation: it shortenedcoagulation time from 140 sec (the time for mouse plasma in CaCl₂ tocoagulate in the absence of added antibodies or TF under the specificconditions used) to 60 sec. In contrast, the control bispecificantibodies did not induce activation of coagulation: in these casescoagulation time was 140 sec.

[0472] Later studies confirmed and extended the initial results. Mouseplasma added to A20 cells to which tTF had been tethered withB21-2/10H10 coagulated rapidly. Fibrin strands were visible 36 secondsafter adding the plasma as compared with 164 seconds in plasma added tountreated A20 cells (Table VII). Only when tTF had been tethered to thecells was coagulation induced: no effect on coagulation time was seenwith cells incubated with of tTF alone, homodimeric F(ab′)₂ Fab′fragments or bispecific antibodies having only one of the twospecificities needed for tethering tTF.

[0473] A linear relationship existed between the logarithm of theaverage number of tTF molecules tethered to each A20 cell and therapidity with which those cells induced coagulation of mouse plasma.Cells bearing 300,000 molecules of tTF per cell induced coagulation in40 secs but even with 20,000 molecules per cell coagulation wassignificantly faster (140 secs) than it was with untreated cells (190secs). TABLE VII Coagulation of mouse plasma induced by tethering tTF toA20 cells with bispecific antibody. Reagents added¹ Coagulation time²(sec) None 164 ± 4 B21-2/10H10 + tTF  36 ± 2 B21-2/10H10 163 ± 2 tTF 163± 3 B21-2/OX7 + tTF 165 ± 4 SFR8/10H10 + tTF 154 ± 5 10H10 F(ab')₂ + tTF160 ± 3 10H10 Fab' + tTF 162 ± 2 B21-2 F(ab')₂ + tTF 168 ± 4 B21-2Fab' + tTF 165 ± 4

EXAMPLE III Specific Tumor Vasculature Specific Coagulation In Vivo

[0474] The present example describes the specific coagulation of tumorvasculature in vivo that results following the administration of thebispecific antibody coaguligand as a delivery vehicle for human tissuefactor.

[0475] A. Materials and Methods

[0476] 1. Reagents

[0477] Mouse blood was obtained by heartpuncture and collected in{fraction (1/10)} volume of 3.8%. buffered citrate. The blood wascentrifuged for 10 min at 3000 g and the plasma snap frozen in smallaliquots and stored at −70° C.

[0478] 2. Animals

[0479] BALB/c nu/nu mice were obtained from Simonsen (Gilroy, Calif.)and maintained under SPF conditions.

[0480] 3. C1300 (Muγ) Mouse Model and Treatment

[0481] The tumor model was as previously described (Burrows et al.,1992; Burrows & Thorpe, 1993) with three refinements. First, a differentantibody, B21-2, was used. This antibody recognizes I-A^(d) but notI-E^(d), unlike the previously used M5/114 antibody which recognizesboth molecules. The B21-2 antibody has an approximately 10-fold betteraffinity than M5/114. Second, a subline of the previously usedC1300(Muγ) 12 line was used which grew continuously in BALB/c nu/numice. The C1300(Muγ) 12 cells used previously had to be mixed withuntransfected C1300 cells in order to form continuously growing tumors.The new subline, designated C1300(Muγ) t1P3, will be referred tohereafter as C1300(Muγ). Third, it was unnecessary to add tetracyclineto the mice's drinking water to prevent gut bacteria from inducingI-A^(d) on the gastrointestinal epithelium. Unlike immunotoxins,coaguligands do not damage I-A^(d)-expressing intestinal epithelium.

[0482] For establishment of solid tumors, 1.5×10⁷ C1300 (Muγ) cells wereinjected subcutaneously into the right anterior flank of BALB/c nu/numice. When the tumors had grown to 0.8 cm in diameter, mice wererandomly assigned to different treatment groups each containing 7-8mice.

[0483] Coaguligands were prepared by mixing bispecific antibodies (140μg) and tTF (110 μg) in a total volume of 2.5 ml of 0.9% NaCl andleaving them at 4° C. for one hour. Mice then received intravenousinjections of 0.25 ml of this mixture (i.e. 14 μg of bispecific antibodyplus 11 μg of tTF). Other mice received 14 μg of bispecific antibodiesor 11 μg of tTF alone. The injections were performed slowly into one ofthe tail veins over approximately 45 sec and were followed with a secondinjection of 200 μl of saline into the same vein. This injectionprocedure was adopted to prevent thrombosis of the tail vein which wasseen if mice were rapidly injected (1-2 sec). Seven days later, thetreatments were repeated.

[0484] Perpendicular tumor diameters were measured at regular intervalsand tumor volumes were estimated according to the following equation:

volume=smaller diameter²×larger diameter×π/6

[0485] Differences in tumor volume were tested for statisticalsignificance using the Mann-Whitney-Wilcoxon nonparametric test for twoindependent samples (Gibbons, 1976).

[0486] For histopathological analyses, animals were anesthetized withmetophane at various times after treatment and were exsanguinated byperfusion with heparinized saline. 500 IU of heparin were i.v. injected,the animal anesthetized with metofane and the systemic circulationperfused with PBS at a flow rate of 0.6 mls/min until the liver had beencleared of blood. The tumor and normal tissues were excised and formalinfixed (4% v/v). Paraffin sections of the tissues were cut and stainedwith the standard Martius Scarlet Blue (MSB) trichrome technique fordetection of fibrin, and with hematoxylin and eosin stain for cellmorphology.

[0487] B. Results

[0488] 1. Improved Tumor Model

[0489] To improve the C1300 (Muγ) tumor model as described before(Burrows et al., 1992), the inventors subcloned the C1300 (Muγ) cellline into a cell line that can grow without being mixed with itsparental cell, C1300, but still express the I-A^(d) MHC Class II antigenon the endothelial cells of the tumor. The inventors used ananti-I-A^(d) antibody (B21-2) that has a 5-10 fold higher affinity forits antigen than the initial anti-I-A^(d) antibody (M5/114.15.2) used inthis model as determined by FACS. In vivo distribution studies with thisnew anti-I-A^(d) antibody showed the same tissue distribution pattern asdid M5/114.15.2. Intense staining with B21-2 was seen in tumor vascularendothelium, light to moderate staining in Kuppfer cells in the liver,the marginal zones in the spleen and some areas in the small and largeintestines. Vessels in other normal tissues were unstained.

[0490] 2. Determination of Suitable In Vivo Doses

[0491] The maximum tolerated dose was 16 μg B21-2/10H10 plus 11 μg tTFinjected intravenously into the tail vein of mice. At this dose, micelost no weight and had normal appearance and activity levels. At ahigher dose of 20 Ag B21-2/10H10 plus 16 μg tTF, two of ten micedeveloped localized dermal hemorrhages which eventually resolved. Thelower dose was adopted for in vivo studies. Truncated TF itself was nottoxic at 50 μg, given intravenously.

[0492] 3. Specific Coagulation and Infarction in Tumor Vasculature

[0493] Intravenous administration of a coaguligand composed ofB21-2/10H10 (20 μg) and tTF (16 μg) to mice bearing solid C1300 (Muγ)tumors caused tumors to assume a blackened, bruised appearance within 30minutes. A histological study of the time course of events within thetumor revealed that 30 minutes after injection of coaguligand allvessels in all regions of the tumor were thrombosed (FIG. 3B). Vesselscontained platelet aggregates, packed red cells and fibrin. At thistime, tumor-cells were healthy, being indistinguishable morphologicallyfrom tumor cells in untreated mice (FIG. 3A).

[0494] By 4 hours, signs of tumor cell distress were evident. Themajority of tumor cells had begun to separate from one another and haddeveloped pyknotic nuclei (FIG. 3C). Erythrocytes were commonly observedin the tumor interstitium. By 24 hours, advanced tumor necrosis wasvisible throughout the tumor (FIG. 3D). By 72 hours, the entire centralregion of the tumor had compacted into morphologically indistinctdebris.

[0495] In one of three of the tumors examined, a viable rim of tumorcells 5-10 cell layers thick was visible on the outskirts of the tumorwhere it was infiltrating into surrounding normal tissues.Immunohistochemical examination of serial sections of the same tumorrevealed that the vessels in the regions of tumor infiltration lackedclass II antigens.

[0496] Tumors from control mice which had received B21-2/10H10bispecific antibody (20 μg) alone 30 minutes or 24 hours earlier showedno signs of infarction. Tumors from mice which received tTF (16 μg),alone or in combination with B21-2/OX7 or SFR8/10H10, showed no signs ofinfarction 30 min after injection but 24 hours after injection,occasional vessels (about 20% of vessels overall) in the tumor wereinfarcted. These appeared to be most prevalent in the core of the tumor.

[0497] No thrombi or morphological abnormalities were visible inparaffin sections of liver, kidney, lung, intestine, heart, brain,adrenals, pancreas and spleen taken from tumor-bearing mice 30 minutes,4 hours and 24 hours after administration of coaguligand or tTF.

[0498] 4. Tumor Regressions of Solid Tumors

[0499]FIG. 4 shows the results of a representative anti-tumor study inwhich a coaguligand composed of B21-2/10H10 and tTF was administered tomice with 0.8 cm diameter tumors. The tumors regressed to approximatelyhalf their pretreatment size. Repeating the treatment on the 7th daycaused the tumors to regress further, usually completely. In 5/7animals, complete regressions were obtained. Two of the micesubsequently relapsed four and six months later. These anti-tumoreffects are statistically highly significant (P<0.001) when comparedwith all other groups.

[0500] Tumors in mice treated with tTF alone or with tTF mixed with theisotype-matched control bispecific antibodies, SFR8/10H10 or B21-2/OX7,grew more slowly than those in groups receiving antibodies or diluentalone. These differences were statistically significant (P<0.05) on days12-14. Thus, part of the anti-tumor effects seen with the B21-2/10H10coaguligand are attributable to a slight non-specific action of tTFitself.

[0501] At the end of the study, two mice which had been treated withdiluent alone and which had very large tumors of 2.0 cm³ and 2.7 cm³(i.e. 10-15% of their body weight) were given coaguligand therapy. Bothhad complete remissions although their tumors later regrew at theoriginal site of tumor growth.

[0502] C. Discussion

[0503] The present studies show that soluble human tTF, possessing.practically no ability to induce coagulation, became a powerfulthrombogen for tumor vasculature when targeted by means of a bispecificantibody to tumor endothelial cells. In vitro coagulation studies showedthat the restoration of thrombotic activity of tTF is mediated throughits cross-linking to antigens on the cell surface.

[0504] tTF binds factors VII and VIIa with high affinity and enhancesthe catalytic activity of VIIa but does not induce coagulation of plasmabecause the tTF:VIIa complex has to be associated with a membranesurface for efficient activation of factors IX and X (Ruf et al., 1991;Krishnaswamy et al., 1992). Tethering of tTF:VIIa to the cell surface bymeans of a bispecific antibody restores its ability to inducecoagulation by bringing the tTF:VIIa into close proximity to themembrane: the membrane phospholipid provides the surface on which thecoagulation-initiation complexes with factors IX or X can assemble andefficiently produce intermediates in the clotting process.

[0505] Administration of a coaguligand directed against class II to micehaving tumors with class II-expressing vasculature caused rapidthrombosis of blood vessels throughout the tumor. This was followed byinfarction of the tumor and complete tumor regressions in a majority ofanimals. In those animals where complete regressions were not obtained,the tumors grew back from a surviving rim of tumor cells on theperiphery of the tumor where it had infiltrated into the surroundingnormal tissues. The vessels at the growing edge of the tumor lackedclass II antigens, thus explaining the lack of thrombosis of thesevessels by the coaguligand. It is likely that these surviving cellswould have been killed by coadministering a drug acting on the tumorcells themselves, as was found previously (Burrows & Thorpe, 1993).

[0506] The anti-tumor effects of the coaguligand were similar inmagnitude to those obtained in the same tumor model with an immunotoxincomposed of anti-class II antibody and deglycosylated ricin A-chain(Burrows & Thorpe, 1993). One difference between the two agents is theirrapidity of action. The coaguligand induced thrombosis of tumor vesselsin less than 30 minutes whereas the immunotoxin took 6 hours to achievethe same effect. The immunotoxin acts more slowly because thrombosis issecondary to endothelial cell damage caused by the shutting down ofprotein syntheses.

[0507] A second and important difference between the immunotoxin and thecoaguligand is that they have different toxic side effects. Theimmunotoxin caused a lethal destruction of class II-expressinggastrointestinal epithelium unless antibiotics were given to suppressclass II induction by intestinal bacteria. The coaguligand caused nogastrointestinal damage, as expected because of the absence of clottingfactors outside of the blood, but caused coagulopathies in occasionalmice when administered at high dosage.

[0508] The findings described in this report demonstrate the therapeuticpotential of targeting human coagulation-inducing proteins to tumorvasculature. For clinical application, antibodies or other ligands areneeded that bind to molecules that are present on the surface ofvascular endothelial cells in solid tumors but absent from endothelialcells in normal tissues. Tumor endothelial markers could be induceddirectly by tumor-derived angiogenesis factors (Folkman, 1985) orcytokines (Burrows et al., 1991; Ruco et al., 1990), or could relate tothe rapid proliferation (Denekamp & Hobson, 1982) and migration(Folkman, 1985) of endothelial cells during neovascularization.

[0509] Several candidate antibodies have been described. The antibodyTEC-11, against endoglin is a particular example that binds selectivelyto human tumor endothelial cells.

[0510] Other antibodies include FB5, against endosialin (Rettig et al.,1992), E-9, against an endoglin-like molecule (Wang et al., 1993), BC-1,against a fibronectin isoform (Carnemolla et al., 1989) and TP-1 andTP-3, against an osteosarcoma-related antigen (Bruland et al., 1988).CD34 has been reported to be upregulated on migrating endothelial cellsand on the abluminal processes of budding capillaries in tumors andfetal tissues (Schlingemann et al., 1990). The receptors for vascularendothelial cell growth factor (VEGF) become upregulated in tumor bloodvessels (Plate et al., 1993; Brown et al., 1993) probably in response tohypoxia (Thieme et al., 1995), and selectively concentrate VEGF in tumorvessels (Dvorak et al., 1991).

[0511] The induction of tumor infarction by targetingcoagulation-inducing proteins to these and other tumor endothelial cellmarkers is proposed as a valuable new approach to the treatment of solidtumors. The coupling of human (or humanized) antibodies to humancoagulation proteins to produce wholly human coaguligands isparticularly contemplated, thus permitting repeated courses of treatmentto be given to combat both the primary tumor and its metastases.

EXAMPLE IV Synthesis of Truncated Tissue Factor (tTF) Constructs

[0512] tTF is herein designated as the extracellular domain of themature tissue factor protein (amino acid 1-219 of the mature protein;SEQ ID NO: 23). SEQ ID NO: 23 is encoded by, e.g., SEQ ID NO: 22.

[0513] A. H₆[tTF]

[0514] H₆ Ala Met Ala [tTF]. The tTF complimentary DNA (cDNA) wasprepared as follows: RNA from J-82 cells (human bladder carcinoma) wasused for the cloning of tTF. Total RNA was isolated using the GlassMax™RNA microisolation reagent (Gibco BRL). The RNA was reverse transcribedto cDNA using the GeneAmp RNA PCR kit (Perkin Elmer). tTF cDNA wasamplified using the same kit with the following two primers: 5′ primer:5′ GTC ATG CCA TGG CCT CAG GCA CTA CAA (SEQ ID NO:1) 3′ Primer: 5′ TGACAA GCT TAT TCT CTG AAT TCC CCC TTT CT (SEQ ID NO:2)

[0515] The underlined sequences codes for the N-terminus of tTF. Therest of the sequence in the 5′ primer is the restriction site for NcoIallowing the cloning of tTF into the expression vector. The sequence inthe 3′ primer is the HindIII site for cloning tTF into the expressionvector. PCR amplification was performed as suggested by themanufacturer. Briefly, 75 μM dNTP; 0.6 μM primer, 1.5 mM MgCl₂ were usedand 30 cycles of 30″ at 95° C., 30″ at 55° C. and 30″ at 72° C. wereperformed.

[0516] The E. coli expression vector H₆ pQE-60 was used for expressingtTF (Lee et al., 1994). The PCR amplified tTF cDNA was inserted betweenthe NcoI and Hind3 site. H₆ pQE-60 has a built-in (His)₆ encodingsequence such that the expressed protein has the sequence of (His)₆ atthe N terminus, which can be purified on a Ni-NTA column.

[0517] To purify tTF, tTF containing H6 pQE-60 DNA was transformed to E.coli TG-1 cells. The cells were grown to OD₆₀₀=0.5 and IPTG was added to30 μM to induce the tTF production. The cells were harvested aftershaking for 18 h at 30° C. The cell pellet was denatured in 6 M Gu-HCland the lysate was loaded onto a Ni-NTA column (Qiagen). The bound tTFwas washed with 6 M urea and tTF was refolded with a gradient of 6 M−1 Murea at room temperature for 16 h. The column was washed with washbuffer (0.05 Na H₂ PO₄, 0.3 M Nacl, 10% glycerol) and tTF was elutedwith 0.2 M Imidozole in wash buffer. The eluted tTF was concentrated andloaded onto a G-75 column. tTF monomers were collected.

[0518] B. tTF

[0519] Gly[tTF]. The GlytTF complimentary DNA (cDNA) was prepared thesame way as described in the previous section except the 5′ primer wasreplaced by the following primer in the PCR.

[0520] 5′ primer: 5′ GTC ATG CCA TGG CCC TGG TGC CTC GTG CTT CTG GCA CTACAA ATA CT (SEQ ID NO: 3)

[0521] The underlined sequence codes for the N-terminus of tTF. Theremaining sequence encodes a restriction site for NcoI and a cleavagesite for thrombin.

[0522] The H₆ pQE60 expression vector and the procedure for proteinpurification is identical to that described above except that the finalprotein product was treated with thrombin to remove the H₆ peptide. Thiswas done by adding 1 part of thrombin (Sigma) to 500 parts of tTF (w/w),and the cleavage was carried out at room temperature for 18 h. Thrombinwas removed from tTF by passage of the mixture through a BenzamidineSepharose 6B thrombin affinity column (Pharmacia).

[0523] C. Cysteine-Modified tTFS

[0524] tTF constructs were modified with an N or C-terminal cysteine toallow for easier conjugation to derivatized antibody through a disulfidebond.

[0525] H₆ C[tTF]. (His)₆ Ala Met Ala Cys-[tTF]. The DNA was made asdescribed in the previous section except that the 5′ primer was replacedby the following primer in the PCR.

[0526] 5′ primer: 5′ GTC ATG CCA TGG CCT GCT CAG GCA CTA CAA ATA CTG TG(SEQ ID NO: 4)

[0527] All of the procedures were the same as described above, exceptthe N-terminal cys was protected with an exchangeable oxidizing/reducingreagent.

[0528] C[tTF]. Gly Ser Cys [tTF2-219]. The DNA was made as described inthe previous section except that the 5′ primer was replaced by thefollowing primer in the PCR.

[0529] 5′ primer: 5′ GTC ATG CCA TGG CCC TGG TGC CTC GTG GTT CTT GCG GCACTA CAA ATA CT (SEQ ID NO: 5)

[0530] The vector construct and protein purification is the same asdescribed for the (His)₆ Ala Met Ala Cys [tTF] construct, except thatthrombin treatment was used to remove the (His)₆ as described above.

[0531] H₆ [tTF]C. (His)₆ Ala Met Ala [tTF] Cys. The DNA was made thesame way as described in the (His)₆ AMA [tTF] sections, except that the3′ primer was replaced by the following primer.

[0532] 3′ primer:5′ TGA CAA GCT TAG CAT TCT CTG AAT TCC CCC TTT CT (SEQID NO: 6)

[0533] The underlined sequence encodes the C-terminus of tTF. The restof the sequence contains the HindIII restriction site for cloning tTF into the expression vector.

[0534] All of the procedures are the same as described in the tTFsection except that 10 mM β-ME was used in the 6 M Gu-HCl denaturingsolution and the C-terminal cysteine was protected with an exchangeableoxidizing/reducing reagent.

[0535] Other [tTF] Cys monomers, such as [tTF 1-220] Cys, [tTF 1-221]Cys and [tTF 1-222] Cys are also made (and conjugated) using the samemethodology.

[0536] D. C Linker [tTF]

[0537] The C Linker [tTF],Gly-Ser-Cys-(Gly)₄-Ser-(Gly)₄-Ser-(Gly)₄-Ser-[tTF], was alsoconstructed. The cDNA was made using a two step PCR procedure asfollows:

[0538] PCR 1: amplification of linker DNA

[0539] cDNA encoding the NcoI site, the thrombin cleavage site,cysteine, linker and the N-terminus of tTF was amplified using thefollowing primers: 5′ primer: 5′GTC ATG CCA TGG CCC TGG TGC CTC GTG GTTGC G (SEQ ID NO:7)            GA GGC GGT GGA TCA GGC 3′ primer: 5′AGTATT TGT AGT GCC TGA GGA TCC GCC ACC TCC (SEQ ID NO:8)            ACT

[0540] The underlined sequences encode the linker peptide. The DNAtemplate used in the PCR was double strand DNA encoding the followinglinker.

[0541] Sequence: GGA GGC GGT GGA TCA GGC GGT GGA GGT AGT GGA GGT GGC GGATCC (SEQ ID NO: 9)

[0542] The same PCR conditions were used as described in the tTFsection. The 95 b.p. amplified product was linked to tTF DNA in thePCR2.

[0543] PCR 2: Linking the Cys-linker DNA to tTF DNA. DNA templates usedin the PCR were two overlapping DNA: The 95 b.p. DNA from PCR 1 asdescribed above and tTF DNA. The primers used were the following:5′ primer: 5′GTC ATG CCA TGG CCC TG (SEQ ID NO:10) 3′ primer: 5′TGA CAAGCT TAT TCT CTG AAT TCC CCC TTT CT (SEQ ID NO:11)

[0544] The final PCR product of 740 b.p. was digested with NcoI andHindIII and inserted into the H6 pQE 60 as described in the tTF section.

[0545] The vector constructs and protein purification procedures are allthe same as described in the C[tTF] section.

EXAMPLE V Synthesis of Dimeric Tissue Factor

[0546] The inventors' reasoned that tissue factor dimers may be morepotent than monomers at initiating coagulation. It is possible thatnative tissue factor on the surface of J82 bladder carcinoma cells mayexist as a dimer (Fair et al., 1987). The binding of one factor VII orVIIa molecule to one tissue factor molecule may also facilitate thebinding of another factor VII or VIIa to another tissue factor (Fair etal., 1987; Bach et al., 1986). Furthermore, tissue factor showsstructural homology to members of the cytokine receptor family(Edgington et al., 1991) some of which dimerize to form active receptors(Davies and Wlodawer, 1995). The inventors therefore synthesized TFdimers, as follows.

[0547] A. [tTF] Linker [tTF].

[0548] The Gly [tTF] Linker [tTF] with the structure Gly[tTF] (Gly)₄ Ser(Gly)₄ Ser (Gly)₄ Ser [tTF] was made. Two pieces of DNA were PCRamplified separately and were ligated and inserted into the vector asfollows:

[0549] PCR 1: Preparation of tTF and the 5′ half of the linker DNA. Theprimer sequences in the PCR are as follows: 5′ primer: 5′ GTC ATG CCATGG CCC TGG TGC CTC GTG GTT CTT GCG GCA CTA CAA ATA CT (SEQ ID NO:12)3′ primer: 5′ CGC GGA TCC ACC GCC ACC AGA TCC ACC GCC TCC TTC TCT GAATTC CCC TTT CT (SEQ ID NO:13)

[0550] Gly[tTF] DNA was used as the DNA template. Further PCR conditionswere as described in the tTF section.

[0551] PCR 2: Preparation of the 3′ half of the linker DNA and tTF DNA.The primer sequences in the PCR were as follows: 5′ primer: 5′ CGC GGATCC GGC GGT GGA GGC TCT TCA GGC ACT ACA AAT ACT GT (SEQ ID NO:14)3′ primer: 5′ TGA CAA GCT TAT TCT CTG AAT TCC CCT TTC T (SEQ ID NO:15)

[0552] tTF DNA was used as the template in the PCR. The product from PCR1 was digested with NcoI and BamH. The product from PCR 2 was digestedwith HindIII and BamH1. The digested PCR1 and PCR2 DNA were ligated withNcoI and HindIII-digested H₆ pQE 60 DNA.

[0553] For the vector constructs and protein purification, theprocedures were the same as described in the Gly [tTF] section.

[0554] B. Cys [tTF] Linker [tTF]

[0555] The Cys [tTF] Linker [tTF] with the structure Ser Gly Cys [tTF2-219] (Gly)₄ Ser (Gly)₄ Ser(Gly)₄ Ser [tTF] was also constructed. DNAwas made by PCR using the following primers were used: 5′ primer: 5′ GTCATG CCA TGG CCC TGG TGC CTC GTG GTT CTT GCG GCA CTA CAA ATA CT (SEQ IDNO:16) 3′ primer: 5′ TGA CAA GCT TAT TCT CTG AAT TCC CCT TTC T (SEQ IDNO:17)

[0556] [tTF] linker [tTF] DNA was used as the template. The remainingPCR conditions were the same as described in the tTF section. The vectorconstructs and protein purification were all as described in thepurification of H₆C[tTF].

[0557] C. [tTF] Linker [tTF]cys

[0558] The [tTF] Linker [tTF]cys dimer with the protein structure [tTF](Gly)₄ Ser (Gly)₄ Ser (Gly)₄ Ser [tTF] Cys was also made. The DNA wasmade by PCR using the following primers: 5′ primer: 5′ GTC ATG CCA TGGCCC TGG TGC CTC GTG GTT GCA CTA CAA ATA CT (SEQ ID NO:18) 3′ primer:5′ TGA CAA GCT TAG CAT TCT CTG AAT TCC CCT TTC T. (SEQ ID NO:19)

[0559] [tTF] linker [tTF] DNA was used as the template. The remainingPCR conditions were the same as described in the tTF section. The vectorconstructs and protein purification were again performed as described inthe purification of [tTF]cys section.

[0560] D. Chemically Conjugated Dimers

[0561] [tTF] Cys monomer are conjugated chemically to form [tTF] Cys-Cys[tTF] dimers. This is done by adding an equal molar amount of DTT to theprotected [tTF] Cys at room temperature for 1 hr to deprotect and exposethe cysteine at the C-terminus of [tTF] Cys. An equal molar amount ofprotected [tTF] Cys is added to the DTT/[tTF] Cys mixture and theincubation is continued for 18 h at room temperature. The dimers arepurified on a G-75 gel filtration column.

[0562] The Cys [tTF] monomer is conjugated chemically to form dimersusing the same method.

EXAMPLE VI Synthesis of Tissue Factor Mutants

[0563] Two tTF mutants are described that lack the capacity to converttTF-bound factor VII to factor VIIa. There is 300-fold less factor VIIain the plasma compared with factor VII (Morrissey et al., 1993).Therefore, circulating mutant tTF should be less able to initiatecoagulation and hence exhibit very low toxicity. In coaguligands, oncethe mutant tTF has localized through the attached antibody to the tumorsite, Factor VIIa will be injected to exchange with the tTF-bound FactorVII. The mutants are active in the presence of factor VIIa.

[0564] A. [tTF]G164A

[0565] The “[tTF]G164A” has the mutant protein structure with the aminoacid 164 (Gly) of tTF being replaced by Ala. The Chameleondouble-stranded site directed mutagenesis kit (Stratagene) is used forgenerating the mutant. The DNA template is Gly[tTF] DNA and the sequenceof the mutagenizing primer is:

[0566] 5° CAA GTT CAG CCA AGA AAAC (SEQ ID NO: 20)

[0567] The vector constructs and protein purification proceduresdescribed above are used in the purification of Gly[tTF].

[0568] B. [tTF] W158R S162A

[0569] The [tTF]W158R S162A is a double mutant in which amino acid 158(Trp) of tTF is replaced by Arg and amino acid 162 (Ser) is replaced byAla. The same mutagenizing method is used as described for [tTF] G164A.The mutagenizing primer is:

[0570] 5′ ACA CTT TAT TAT CGG AAA TCT TCA GCT TCA GGA AAG (SEQ ID NO:21)

[0571] The foregoing vector constructs and protein purificationprocedures are used for purifying Gly[tTF].

EXAMPLE VII Synthesis of Tissue Factor Conjugate

[0572] A. Chemical Derivatization and Antibody Conjugation

[0573] Antibody tTF conjugates were synthesized by the linkage ofchemically derivatized antibody to chemically derivatized tTF via adisulfide bond (as exemplified in FIG. 5).

[0574] Antibody was reacted with a 5-fold molar excess of succinimidyloxycarbonyl-α-methyl α-(2-pyridyldithio)toluene (SMPT) for 1 hour atroom temperature to yield a derivatized antibody with an average of 2pyridyldisulphide groups per antibody molecule. Derivatized antibody waspurified by gel permeation chromatography.

[0575] A 2.5-fold molar excess of tTF over antibody was reacted with a45-fold molar excess of 2-iminothiolane (2 IT) for 1 hour at roomtemperature to yield tTF with an average of 1.5 sulfhydryl groups pertTF molecule. Derivatized tTF was also purified by gel permeationchromatography and immediately mixed with the derivatized antibody.

[0576] The mixture was left to react for 72 hours at room temperatureand then applied to a Sephacryl S-300 column to separate theantibody-tTF conjugate from free tTF and released pyridine-2-thione. Theconjugate was separated from free antibody by affinity chromatography ona anti-tTF column. The predominant molecular species of the finalconjugate product was the singly substituted antibody-tTF conjugate (Mrapprox. 176,000) with lesser amounts of multiply substituted conjugates(Mr≧approx. 202,000) as assessed by SDS-PAGE.

[0577] B. Conjugation of Cysteine-Modified tTF to Derivatized Antibody

[0578] Antibody-C[TF] and [tTF]C conjugates were synthesized by directcoupling of cysteine-modified tTF to chemically derivatized antibody viaa disulfide bond (as exemplified in FIG. 5).

[0579] Antibody was reacted with a 12-fold molar excess of 2IT for 1hour at room temperature to yield derivatized antibody with an averageof 1.5 sulfhydryl groups per antibody molecule. Derivatized antibody waspurified by gel permeation chromatography and immediately mixed with a2-fold molar excess of cysteine-modified tTF. The mixture was left toreact for 24 hours at room temperature and then the conjugate waspurified by gel permeation and affinity chromatography as describedabove.

[0580] The predominant molecular species of the final conjugate was thesingly substituted conjugate (Mr approx. 176,000) with lesser amounts ofmultiple substituted conjugates (Mr≧approx. 202,000) as assessed bySDS-PAGE.

[0581] C. Conjugation of Cysteine-Modified tTF to Fab′ Fragments

[0582] Antibody Fab′-C[tTF] and [tTF]C conjugates are prepared. Suchconjugates may be more potent in vivo because they should remain on thecell surface for longer than bivalent conjugates due to their limitedinternalization capacity. Fab′ fragments are mixed with a 2-fold molarexcess of cysteine-modified tTF for 24 hours and then the conjugatepurified by gel permeation and affinity chromatography as describedabove.

[0583] D. Clotting Activity of tTF Conjugates

[0584] tTF conjugates were prepared with the B21-2 monoclonal antibodywhich binds to Class II antigens expressed on the surface to A20 cells.The conjugates were prepared with chemically derivatized tTF andcysteine-modified tTF and the ability of the conjugates to clot mouseplasma in CaCl₂ was determined after their binding to the surface of A20cells.

[0585] Both B21-2 conjugates shortened the clotting time of mouse plasmain CaCl₂ (control) in a dose-dependent manner. The tTF conjugatesdisplayed a similar enhancement in coagulation as occurred when tTF wastethered to the surface of A20 cells with the bispecific antibodyB21-2/10H10 (FIG. 6).

[0586] E. Anti-tumor Cell tTF Conjugates

[0587] It has already been established that when tTF is targeted totumor vascular endothelial cells it induces coagulation within the tumorvessels (Examples I through III). The inventors' contemplated thatcoagulation would be induced in tumor vessels if tTF was targeted to thesurface of tumor cells.

[0588] Three anti-tumor cell antibodies, KS1/4, D612, and XMMCO-791,were conjugated to tTF as described in the “Preparation of tTFconjugates” section above. KS1/4 was obtained from Dr. R. Reisfeld atthe Scripps Research Institute, Department of Immunology, La Jolla,Calif., and is also described in U.S. Pat. No. 4,975,369; D612 wasobtained from Dr. J. Schlom at the NCl, Laboratory of Tumor Immunologyand Biology, Bethesda, Maryland, is described in U.S. Pat. No. 5,183,756and can be obtained from culture supernatants from the ATCC hybridomacell line Accession No. HB 9796; XMMCO-791 was purified from tissueculture supernatant from the hybridoma cell line purchased from theATCC.

[0589] The human colon carcinoma cell line Widr was used as a targetcell for KS1/4. Widr cells were purchased from the ATCC and weremaintained in DMEM supplemented with 10% (v/v) fetal calf serum,L-glutamine and antibiotics in an atmosphere of 10% (v/v) CO₂ in air.The human colon carcinoma cell line LS147T was used as a target cell forD612. LS147T cells were purchased from the ATCC and were maintained inRPMI supplemented with 10% (v/v) fetal calf serum, L-glutamine andantibiotics in an atmosphere of 5% (v/v) CO2 in air. The human non smallcell lung cancer cell line H460 was used as a target cell for XMMCO-791.H460 cells were obtained from Dr. Adi Gazdar, Simmons Cancer Center,University of Texas Southwestern Medical Center, Dallas, Tex. and weremaintained in DMEM supplemented with 10% (v/v) fetal calf serum,L-glutamine and antibiotics in an atmosphere of 10t (v/v) CO₂ in air.All three cell lines grew as adherent monolayers.

[0590] The conjugates were tested for their ability to enhance theclotting time of mouse plasma in CaCl₂ when bound to tumor cellsexpressing the relevant target antigens. Tumor cells were removed fromtissue culture flasks with 0.05% (w/v) EDTA in PBS. The cells werepreincubated with TF9-6B4 and TF8-5G9 antibodies to neutralize anynative tissue factor activity (Morrisey et. al., 1988) and then thecoagulation assay was performed as described for A20 cells.

[0591] When bound to their target cell line, all three conjugatesshortened the clotting time of mouse plasma in CaCl₂ (control) in adose-dependent manner (FIG. 7), indicating that coagulation wasaccelerated at the surface of tumor cells when tTF was targeted to thecell surface.

EXAMPLE VIII Synthesis of Tissue Factor Prodrugs

[0592] Exemplary tTF prodrugs have the following structures: tTF₁₋₂₁₉(X)_(n1) (Y)_(n2) Z Ligand, where tTF₁₋₂₁₉ represents TF minus thecytosolic and transmembrane domains; X represents a hydrophobictransmembrane domain n1 amino acids (AA) in length (1-20 AA); Yrepresents a hydrophilic protease recognition sequence of n2 AA inlength (sufficient AA to ensure appropriate protease recognition); Zrepresents a disulfide thioester or other linking group such as(Cys)₁₋₂; Ligand represents an antibody or other targeting moietyrecognizing tumor-cells, tumor EC, connective tissue (stroma) or basallamina markers

[0593] The tTF prodrug is contemplated for injection intravenouslyallowing it to localize to diseased tissue (i.e. tumor). Once localizedin the diseased tissue, endogenous proteases (i.e., metalloproteinases,thrombin, factor Xa, factor VIIa, factor IXa, plasmin) will cleave thehydrophilic protease recognition sequence from the prodrug which willallow the hydrophobic transmembrane sequence to insert into a local cellmembrane. Once the tail has inserted into the membrane, the tTF willregain its coagulation-inducing properties resulting in clot formationin the vasculature of the diseased tissue.

EXAMPLE IX Synthesis of Coagulation Factors Lacking the Gla Modification

[0594] The vitamin-K-dependent coagulation factors (Factor II/IIa,Factor VII/VIIa, Factor IX/IXa and Factor X/Xa) lacking the Gla(γ-carboxyglutamic acid) modification are contemplated to be useful forthe formation of coaguligands. Coagulation factors lacking the Glamodification are poor coagulants because the unmodified factorsassociate inefficiently with lipid membranes: targeting the factor via aligand to the vasculature of tumors or other sites should bring thefactor back into proximity to the cell surface and enable coagulation toproceed in that site.

[0595] “Gla” is made post-translationally by modifying the existing Glu(Glutamic acid) residues. Vitamin-K-dependent coagulation factors(Factor II/IIa, Factor VII/VIIa, Factor IX/IXa and Factor X/Xa) lackingthe Gla modification may be made by expressing the genes that encodethem in a host, such as bacteria, that does not modify Glu to Gla. TheDNA sequences encoding each of Factor II/IIa, Factor VII/VIIa, FactorIX/IXa and Factor X/Xa are included herein as SEQ ID NO: 24, SEQ ID NO:25, SEQ ID NO: 26 and SEQ ID NO: 27, respectively. Procaryoticexpression is therefore straightforward.

[0596] Such Gla-lacking factors may also be made by mutating any of thesequences described above (SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26and SEQ ID NO: 27) to change the corresponding Glu residues to anotheramino acid before expressing the genes, this time in virtually any hostcell. The codon to be changed is the GAG codon (GAA also encodes Glu andis to be avoided). Using Factor VII as an example, the Gla “domain” islocated generally in the 216-325 region. The first Gla-encoding tripletoccurs at 231 of SEQ ID NO: 25, and the last extends through 318 of SEQID NO: 25. The GAG codons may readily be changed using molecularbiological techniques.

[0597]FIG. 8 shows that the Gla domains of each of the abovevitamin-K-dependent coagulation factors lie in an analogous region.Therefore, mutation of the so-called “corresponding” Glu residues in anyone of SEQ ID NO: 24, SEQ ID NO: 26 and SEQ ID NO: 27 will also bestraightforward.

[0598] The following Table of codons is provided to enable readymutation choices to be made in modifying a given Gla-encoding codon orsequence. Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine CysC UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAUProline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGAAGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr TACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAU

[0599] Site-specific mutagenesis is the technique contemplated for usein the preparation of individual vitamin-K-dependent coagulation factorslacking the Gla modification, through specific mutagenesis of theunderlying DNA and the introduction of one or more nucleotide sequencechanges into the DNA.

[0600] Site-specific mutagenesis allows the production of mutantsthrough the use of specific oligonucleotide sequences which encode theDNA sequence of the desired mutation, as well as a sufficient number ofadjacent nucleotides, to provide a primer sequence of sufficient sizeand sequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Typically, a primer of about 17 to 25nucleotides in length is preferred, with about 5 to 10 residues on bothsides of the junction of the sequence being altered.

[0601] In general, the technique of site-specific mutagenesis is wellknown in the art, as exemplified by publications such as Adelman et al.(1983) and by the TF mutant studies described above. The techniquetypically employs a phage vector which exists in both a single strandedand double stranded form. Typical vectors useful in site-directedmutagenesis include vectors such as the M13 phage (Messing et al.,1981). These phage are readily commercially available and their use isgenerally well known to those skilled in the art. Double strandedplasmids are also routinely employed in site directed mutagenesis whicheliminates the step of transferring the gene of interest from a plasmidto a phage.

[0602] In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector or melting apartthe two strands of a double stranded vector which includes within itssequence a DNA sequence which encodes the vitamin-K-dependentcoagulation factor. An oligonucleotide primer bearing the desiredmutated sequence is prepared, generally synthetically, for example bythe method of Crea et al. (1978). This primer is then annealed with thesingle-stranded vector, and subjected to DNA polymerizing enzymes suchas E. coli polymerase I Klenow fragment, in order to complete thesynthesis of the mutation-bearing strand. Thus, a heteroduplex is formedwherein one strand encodes the original non-mutated sequence and thesecond strand bears the desired mutation. This heteroduplex vector isthen used to transform appropriate cells, such as E. coli cells, andclones are selected which include recombinant vectors bearing themutated sequence arrangement.

EXAMPLE X Further Anti-Tumor Vasculature Antibodies

[0603] This example describes the generation of antibodies directedagainst tumor-derived endothelial cell “binding factors” for use indistinguishing between tumor vasculature and the vasculature of normaltissues. Particularly described is the generation of antibodies directedagainst vascular permeability factor (VPF), also termed vascularendothelial cell growth factor (VEGF), and against bFGF (basicfibroblast growth factor).

[0604] For further details concerning FGF one may refer to Gomez-Pinillaand Cotman (1992); Nishikawa et al. (1992), that describe thelocalization of basic fibroblast growth factor; Xu et al. (1992), thatrelates to the expression and immunochemical analysis of FGF; Reilly etal. (1989), that concerns monoclonal antibodies; Dixon et al. (1989),that relates to FGF detection and characterization; Matsuzaki et al.(1989), that concerns monoclonal antibodies against heparin-bindinggrowth factor; and Herbin and Gross (1992), that discuss the bindingsites for bFGF on solid tumors associated with the vasculature.

[0605] In the present studies, rabbits were hyperimmunized withN-terminal peptides of human VEGF, mouse VEGF, guinea pig VEGF, humanbFGF, mouse bFGF or guinea pig bFGF coupled to tuberculin (purifiedprotein derivative, PPD) or thyroglobulin carriers. The peptides were 25to 26 amino acids in length and were synthesized on a peptidesynthesizer with cysteine as the C-terminal residue. Antisera wereaffinity purified on columns of the peptides coupled to Sephraosematrices.

[0606] Antibodies to VEGF were identified by ELISA and by their stainingpatterns on frozen sections of guinea pig tumors and normal tissues.Polyclonal antibodies to guinea pig VEGF and human VEGF reacted with themajority of vascular endothelial cells on frozen sections of guinea pigL10 tumors and a variety of human tumors (parotid, ovarian, mammarycarcinomas) respectively. The anti-human VEGF antibody stained mesangialcells surrounding the endothelial cells in normal human kidneyglomerulae and endothelial cells in the liver, but did not stain bloodvessels in normal human stomach, leg muscle and spleen. The anti-guineapig VEGF antibody did not stain endothelial cells in any normal tissues,including kidney, brain, spleen, heart, seminal vesicle, lung, largeintestine, thymus, prostrate, liver, testicle and skeletal muscle.

[0607] Polyclonal antibodies to human FGF stained endothelial cells inparotid and ovarian carcinomas, but not those in mammary carcinomas.Anti-human FGF antibodies stained glomerular endothelial cells in humankidney, but not endothelial cells in normal stomach, leg muscle andspleen.

[0608] Monoclonal antibodies to guinea pig VEGF, human VEGF and guineapig bFGF were prepared by immunizing BALB/c mice with the N-terminalsequence peptides (with cysteine at the C-terminus of the peptide)coupled to PPD or to thyroglobulin. The synthetic peptides immunogens ofdefined sequence are shown below and are represented by SEQ ID NO: 30,SEQ ID NO: 31 AND SEQ ID NO: 32, respectively: guinea A P M A E G E Q KP R E V V K F M D V Y K R S Y C pig VEGF human A P M A E G G G Q N H H EV V K F M D V Y Q R S Y C VEGF guinea M A A G S I T T L P A L P E G G DG G A F A P G C pig bFGF

[0609] The peptides were conjugated to thyroglobulin or to PPD byderivatizing the thyroglobulin with succimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and reacting thederivative with the peptide. This yields a conjugate having one or morepeptide sequences linked via a thioether bond to thyroglobulin.

[0610] Specifically, the generation of monoclonal antibodies against theabove sequences was achieved using the-following procedure: BALB/c micewere immunized by serial injections with peptide-PPD orpeptide-thyroglobulin into several sites. Four or five days after thelast injection, the spleens were removed and splenocytes were fused withP3xG3Ag8.653 myeloma cells using polyethyleneglycol according to theprocedures published in Morrow, et al. (1991).

[0611] Individual hybridoma supernatants were screened as follows: Firstscreen: ELISA on peptide-thyroglobulin-coated plates. Second screen:ELISA on cysteine linked via SMCC to thyroglobulin. Third screen:Indirect immunoperoxidase staining of frozen sections of guinea pig line10 tumor or human parotid carcinoma. Fourth screen: Indirectimmunoperoxidase staining of frozen sections of miscellaneous malignantand normal guinea pig and human tissues.

[0612] Antibodies were selected that bound to peptide-thyroglobulin butnot to cysteine-thyroglobulin, and which bound to endothelial cells inmalignant tumors more strongly than they did to endothelial cells innormal tissues (Table VIII). TABLE VIII Reactivity of MonoclonalAntibodies Reactivity with Tumour Tumour Endothelium Reactivity MoABImmunogen⁺ Class g. pig human Pattern* GV14 gp VEGF IgM + + BV + sometumor cells GV35 gp VEGF IgM ± ± Tumor cells, weak on BV GV39 gp VEGFIgM + + BV and some tumor cells GV59 gp VEGF IgM + + BV and some tumorcells GV97 gp VEGF IgM + + BV, weak on tumor cells HV55 hu VEGF IgG ? +Basement membrane, some BV GF67 gp FGF IgM + + BV and tumor cells GF82gp FGF IgM + + BV and tumor cells

[0613] A. GV97 Staining of Human and Guinea Pig Tissue Sections

[0614] The GV97 antibody against guinea pig VEGF N-terminus bound toendothelial cells in miscellaneous human malignant (Table IX) and normal(Table X) tissues.

[0615] Binding to endothelial cells in malignant tumors tended to exceedthat to endothelial cells in normal tissues.

[0616] The staining of endothelial cells in guinea pig tumor (line 10hepatocellular carcinoma) and normal tissues was similar in distributionand intensity to that observed with human tissues (Table XI).

[0617] In the Tables, + indicates a positive, as opposed to a negative,result. The numbers 2+, 3+and 4+ refer to a positive signal ofincreasing strength, as is routinely understood in this field of study.TABLE IX Anti-GPVEGF on Human Tumors Purified GV97 1 ug/ml or GV97 GV59Tumor TISSUE 20 ug/ml 10 ug/ml 5 ug/ml 2 ug/ml 0.5 ug/ml supt. GV14 GV39supt. DIGESTIVE TRACT 92-01-A073 2+ 1+ +/− −ve 4+ 4+ esophagus carcinomaM4 Parotid 4+ 87-07-A134 Parotid 3+ 2+ +/− −ve 3+ 4+ carcinoma M5Parotid 4+ 88-04-A010 parotid 1-2+ 1+ −ve −ve 1-3+ adenoca. 90-11-B319Adeno. Ca. 3-4+ 3-4+ of colon to liver 94-02-B021C 3-4+ 3-4+Adenocarcinoma of colon 93-10-A333 Adeno. Ca. 4+ 2-4+ 1-4+ −ve-1+ 4+ 3+of colon with normal 93-02-B004 Villous and 4+ 3-4+ 2-4+ 1-2+ 3-4+ 2-3+Adenomatous polyp of colon 93-02-A130 3+ 2+ +/−-1+ −ve 4+ 4+ 3-4+Leiomyosarcoma in colon 93-02-B020 Gastric Ca. 4+ 2+ 2-3+ −ve-1+ 1-2+ 4+93-04-A221 Pancreas 3-4+ 2-3+ 1-2+ −ve-0.5+ 4+ 4+ Adenoca. 94-04-A390rectum 4+ 3+ 1-2+ 1+ 3+ adenoca. 93-12-A160 tongue 1-2+ +/− −ve −ve 3+3+ carcinomaadenoca. 101-84a Stomach signet 3+ 2+ −ve-1+ −ve most 1-2+3+ ring Ca. (101-84b but a pair) few 3-4+ 90-05-A172 Stomach 4+ 3+ 1-2+−ve-1+ −ve 3+ Adenoca. REPRODUCTIVE TRACT 91-10-A115 Squam. cell 1-4+1-3+ 1-2+ 1-2+ 1-4+ 1-3+ Ca. of vulva 93-03-A343 Prostate +/−-3-4+ +/−to 2-3+ +/− to 1-2+ +/− 3-4+ 3-4+ Adenoca. MUSCLE IMMUNE SYSTEM URINARYSYSTEM 93-10-B002 Renal cell 2+ 3+ Ca. 90-01-A225 Renal cell 4+ 4+ 3-4+of most 1-3+ of 3-4+ 3+ 3-4+ Ca. some 93-01-A257 Transit. 3-4+ 2-3+ 1-2++/− 2-3+ 2-3+ cell Ca. of bladder ENDOCRINE SYSTEM 94-01-A246 4+ 4+ 3-4+3+ 4+ 3-4+ Pheochromocytoma of adrenal 93-11-A074 Adrenal 3-4+ 3-4+ 2-3+1+ 3-4+ 4+ Cort. Ca. RESPIRATORY SYSTEM 93-08-N009 Lung 3-4+ 3-4+ 3-4+Adenoca. 92-10-A316 Sq. cell 4+ 3-4+ 1-2+ −ve-0.5+ 4+ 4+ lung Ca.03-05-A065 Lung 4+ 3-4+ −ve-1+ 1+ 3+ 3+ adenoca. CENTRAL NERVOUS SYSTEM94-01-A299 malig. 4+ 4+ 4+ 3-4+ 4+ 3-4+ metast. schwanoma to Lymph node92-10-A139 Meningioma 4+ 3-4+ 2-3+ 1-2+ 4+ 3-4+ 91-12-A013 Meningioma 4+2-3+ −ve-3+ +/− 4+ 3+ 93-03-A361 Atypical 4+ 4+ 3+ 2+ 4+ 3+ meningiomaINTEGUMENTARY SYSTEM 94-04-V037 Skin Sq. −ve to −ve to 3+ −ve to 1+ −ve2-3+ 2-3+ cell Ca. w/normal 4+ 89-02-225 leg sarcoma 4+ 3-4+ 1+ 1+ 4+ 2+MISC. TUMORS

[0618] TABLE X Anti-GPVEGF on Human Normal Tissues Purified GV97 1 ug/mlor GV59 Tumor TISSUE 20 ug/ml 10 ug/ml 5 ug/ml 2 ug/ml 0.5 ug/ml GV97supt. GV14 GV39 supt. DIGESTIVE SYSTEM 91-01-A128 3+ 2+ 1+ −ve 2-3+ 2-3+Bladder w/ cystitis 94-02-B020 2-3+ 2-3+ uninvolved colon 92-01-A292 N.4+ 4+ 4+ 3-4+ 4+ 3-4+ Colon 93-10-A116 N. Z-4+ 1-4+ 1-3+ −ve-2+ −ve 3-4+2-3+ 3-4+ Colon 90-06-A116 N. 3+ of many 2+ colon 93-02-A350 N. 3-4+ 3+1+ +/− 4+ 4+ esophagus 93-05-A503 N. 4+ 4+ Ileum 94-03-A244 N. 4+ 1-3+−ve-1+ −ve 4+ 4+ Liver 90-02-B132 N. 1+ of a +/− −ve −ve −ve 1-3+ 2-3+2-3+ 2-3+ Liver few 94-01-A181 N. 1-4+ 1-3+ 1-3+ of a −ve 3-4+ Pancreasfew 90-05-D008 N. 2-4+ 1-3+ +/− −ve 2-3+ 2-3+ Pancreas 93-05-A174 N. 2+of a few 1-2+ of a 1+ of a few −ve −ve 3+ of a 2-3+ Parotid few few94-04-A391 N. 1-3+ −ve-2+ −ve −ve 3+ Small bowel 88-06-107 N. 3+ 2+ +/−−ve 3-4+ 3+ Stomach 101-84b N. 3-4+ in main 2-3+ in +/− in main −ve inmain 3+ 3-4+ Stomach (101=84a and main and and 2+ in and 1+ in pair)periphery 3-4+ in periphery periphery periphery 90-11-B337 N. 2-3++/−-1+ −ve −ve 3+ 3+ Stomach REPRODUCTIVE TRACT 93-04-A041 N. 4+ 3+Breast 94-02-A197 N. 4+ 3+ Breast w/fibrocystic change 93-02-A051 Breast−ve-1+ −ve −ve −ve +/− +/−-2+ w/fibrocystic change 93-02-A103 Breast 4+3+ 2+ 1+ w/fibrocyst. change 92-11-A006 N. 2+ of most 1-2+ of most0.5+   −ve −ve 1-2+ of 3+ of ectocervix some most 91-03-A207 N. 2.5+  1.5+   1+ .5+ 2-3+ ectocervix 92-02-A139 N. 1+ in most −ve in most −ve−ve −ve in −ve in ovary w/corp. but 2+ in but 1+ in most but mostlusteum one area one area 3-4+ in bet 3-4 one area in one area93-06-A11B N. 1+ of a few −ve −ve −ve 3+ Prostate 93-11-A317d   3-4+2-3+ −ve-3+ −ve-1+ 3-4+ 3-4+ Prostate chip 93-02-A315 0.5-1+ 0.5+   −ve−ve 1+ 1.2+   Seminal Vesicle 92-04-A069 N. 1+ +/− +/− +/− 1-2+ testis91-04-A117 Ureter 1+ +/− −ve −ve +/−-1+ 3-4+ w/inflammation MUSCLE94-01-A065 N. 3-4+ 2+ +/− −ve 3-4+ 4+ Heart 91-07-D007 N. 1-4+ 1-3+ 1-2+−ve −ve 1-3+ 1-3+ skeletal muscle 95-03-A395 N. 4+ 3-4+ 1-2+ 0.5-1+ 4+4+ Skeletal muscle IMMUNE SYSTEM 90-01-A077 N. 2-3+ 2+ 1+ of some −ve−ve 2-3+ 3-4+ lymph node 90-08-A022 N. most 1+ but most 0,5+ most −vebut most −ve 3+ 3+ lymph node a few 4+ but a few a few 2+ but a few 2+0.5-1+ 91-03-A057 N. 2+ 1+ +/− −ve 3-4+ 3-4+ lymph node 91-09-B017E 3+2+ +/−-1+ −ve 2-3+ 2-3+ uninvolved lymph node 93-07-A236 N. 3-4+ 3-4+−ve-3+ −ve 2-4+ Spicen 93-07-252 N. 3+ 1+ +/− −ve 2-3+ spicen ENDOCRINESYSTEM 94-04-A252 N. 4+ 4+ 3-4+ 1-2+ 4+ 3+ adrenal w/ medulia and cortex93-05-A086 N. most −ve a most −ve a −ve −ve 2-3+ 3-4+ Adrenal medullafew 1-2+ few 1-2+ 92-03-A157 1+ +/− +/− −ve 4+ 4+ Hyperplasic thyroid91-03-B019 N. −ve-3+ −ve-2+ −ve-1+ −ve 2-3+ 2-3+ Thyroid URINARY SYSTEM93-09-A048 N. 4+ 2-3+ Kidney 91-11-A075 N. 4+ 3+ 2+ 1+ 4+ on 4+ on 4+ onKidney glomeruli gbomeruli glomeruli 93-10-B001 N. 4+ 3+ +/− −ve 4+ on4+ on 4+ on Kidney glomeruli glomeruli glomeruli INTEGUMENTARY SYSTEM92-08-A029 N. +/− to 4+ +/− to 3+ +/− to 1+ +/− 2+ 2+ Breast skin89-02-257 4+ 3-4+ 2-3+ 1-2+ 1+ 3-4+ Cartiledge marches 2SS RESPIRATORYSYSTEM 93-05-A203 N. −ve-2+ −ve-1+ +/− −ve 2+ 3+ Lung 92-12-A263 N. 2-3+w/ducts 1-2+ w/ −ve −ve 2-3+ Bronchus staining 3-4+ ducts staining 2-3+

[0619] TABLE XI Staining Pattern of 9F7 anti-VEGF by directimmunohistochemical staining on 6-8 week old GP tissues Purified GV97 1ug/ml or TISSUE 20 ug/ml 10 ug/ml 5 ug/ml 2 ug/ml 0.5 ug/ml 9F7 supt.3F9 supt. 5F9 supt. DIGESTIVE SYSTEM LIVER 2+ 1-2+ +/− +/− 1-2+ 1-2+INTESTINE 4+ 3+ 2+ 1+ 4+m 4+m lymphoid, lymphoid, rest diff. rest diff.than than PANCREAS 1+ of many and 3+ in islands of cells SMALL 4+ ofmany 2-3+ of many 1-2+ of +/− of many 3+ of some 3+ of some INTESTINEand 4+ in and 4+ in many and 4+ and 4+ in and 4+ in and 4+ in lymphoidlymphoid, in lymphoid, lymphoid lymphoid rest diff. lymphoid, rest diff.than fVIII rest diff. than fVIII than fVIII STOMACH 3-4+ 1-2+ on most+/− on most +/− on most 3-4+ (some 3-4+ (some occasional occasionaloccasional fVIII−ve) fVIII−ve) 3+ 2+ 1+ REPRODUCTIVE SYSTEM TESTISMUSCLE AND INTEGUMENTARY SYSTEM HEART −ve −ve −ve −ve 3-4+ (some 3-4+(some fVIII−ve) fVIII−ve) MUSCLE SKIN 1-2+ in 1+ in fatty +/− in +/− infatty 3+ 3+ fatty layer and 3-4+ fatty layer layer and 1-2+ layer and inand 3-4+ of of a few 3-4+ in cellular a few in in cellular cellularlayer cellular layer layer layer IMMUNE SYSTEM SPLEEN 4+ 3+ 2+ −ve 4+ 4+THYMUS URINARY SYSTEM KIDNEY glomeruli glomeruli 3-4+ glomeruliglomeruli 1-2+ glomeruli glomeruli 4+ 2-3+ 3-4+ 3-4+ ENDOCRINE SYSTEMADRENAL RESPIRATORY SYSTEM LUNG NERVOUS SYSTEM CEREBELLUM 4+ 2+ +/− ofmost +/− of most 4+ 4+ and 1+ of a and 1+ of a few few TUMORS TUMOR 4+4+ 3-4+ 2-3+ (2) 4+ 3+

[0620] B. Lack of Reactivity of GV97 With Soluble Human VEGF

[0621] To identify antibodies that are specific for VEGF, the VEGFreceptor (Flk-1) or VEGF bound (or complexed) to the receptor, an ELISAscreening protocol was developed. The procedure is as follows:

[0622] Initially, a 96 well ELISA plate (round bottom) was coated(outside wells left blank) with 100 μl/well of FLK/seap at 10 μg/ml insensitizing buffer. After overnight incubation, the plate was washedtwice with PBS overnight at 4° C. Next the FLK/Seap coated plate wasblocked with 250 μl/well of PBS+CAH (5%) solution for 1 h at 37° C. Theblocking solution was removed and the plate was vigorously tapped onpaper towels.

[0623] The blocked plates were then incubated with 100 μl/well ofVEGF-165 (VEGF 165 aa form produced in yeast obtained from Dr.Ramakrishnan, University of Minnesota) at 2 μg/ml in binding plus 0.1μg/ml heparin for 4 h at room temperature or overnight at 4° C. The VEGFsolution was collected and the plate washed 2 times with PBS-tween(0.10%). Next, 100 μl/well of hybridoma fusion supernatant was added tothe wells and incubated for 1 h at 32° C. Following this supernatantincubation, the plate was washed 3 times with PBS tween and thenincubated with 100 μl well of secondary antibody (KPL, Gt anti-mouse IgGat 1:1000 in PBS tween+CAH (5%) for 1 hour at 37° C.

[0624] Following secondary antibody incubation, the plates were washed 4times with PBS tween, incubated with 100 μl/well of substrate (SubstrateSigma OPD dissolved in citrate buffer+H₂O₂) for 20 minutes and read at490 nm on a Cambridge Technology Microplate Reader (Model 7520). Wellswith an absorbance above appropriate control wells were selected aspositives and further characterized.

[0625] It was found that GV97 did not bind to recombinant VEGF-coatedELISA plates, nor did recombinant human VEGF bind to GV97 coated ELISAplates. Soluble recombinant human VEGF did not block the binding of 5μg/ml GV97 to tumor endothelium in histological sections even when addedat 50 μg/ml.

[0626] These data suggest that GV97 recognizes an epitope of VEGF thatis concealed in recombinant human VEGF but which becomes accessible whenVEGF binds to its receptor on endothelial cells.

[0627] C. GV97 Localization in Line 10-Bearing Guinea Pigs

[0628] In contrast with staining data obtained from histologicalsections, GV97 antibody localized selectively to tumor endothelial cellsafter injection into line 10 tumor-bearing guinea pigs (see Table).Staining of endothelial cells in the tumor was moderately strong whereasstaining of normal endothelium in miscellaneous organs was undetectable.

[0629] D. Anti-bFGF Selectively Bind to Tumor Endothelial Cells

[0630] GV97 and GF82, which had been raised against guinea pig bFGFN-terminus, bound strongly to endothelial cells in frozen reactions ofguinea pig line 10 tumor and to endothelial cells in two types of humanmalignant tumors (see Table XII). By contrast, relatively weak stainingof endothelial cells in miscellaneous guinea pig normal tissues wasobserved. TABLE XII GV97 injected into tumor bearing GP GV 97 20 ug/mlserum volume TISSUE GV97 10 ug/ml injected DIGESTIVE SYSTEM LIVER 2+ −veINTESTINE 3+ possible 0.5-1+ of a few PANCREAS +/− of many and 2+ inpossible 0.5-1+ islands of cells of a few SMALL 2-3+ of many and 4+ in+/− INTESTINE lymphoid, rest diff. than fVIII STOMACH 1-2+ on mostoccasional possibly 0.5+ 3+ of a few REPRODUCTIVE SYSTEM TESTIS +/−MUSCLE AND INTEGUMENTARY SYSTEM HEART −ve −ve MUSCLE −ve SKIN 1+ infatty layer and 3-4+ in cellular layer IMMUNE SYSTEM SPLEEN 3+ Possiblya few 1+ THYMUS URINARY SYSTEM KIDNEY glomeruli 3-4+ ENDOCRINE SYSTEMADRENAL 4+ −ve RESPIRATORY SYSTEM LUNG 2+ −ve NERVOUS SYSTEM CEREBELLUM2+ −ve TUMORS TUMOR 4+ 2-3+

[0631] TABLE XIII Anti-GP FGF Antibody Staining on GP Tissues GP TISSUEGF 67 GF 82 DIGESTIVE SYSTEM LIVER ND ND INTESTINE +/− +/− PANCREAS 2-3+2+ SMALL INTESTINE +/− +/− STOMACH ND ND REPRODUCTIVE SYSTEM TESTIS NDND MUSCLE AND INTEGUMENTARY SYSTEM HEART 2-3+ 1+ MUSCLE +/− 1+ SKIN NDND IMMUNE SYSTEM SPLEEN 3+ −ve THYMUS URINARY SYSTEM KIDNEY 1-2+ −veENDOCRINE SYSTEM ADRENAL 1-2+ +/− RESPIRATORY SYSTEM LUNG 1-2+ 2-3+NERVOUS SYSTEM CEREBELLUM 1+ −1+   TUMORS LINE 1 TUMOR 4+ 4+ HUMANTUMORS PHEOCHROMO CYTOMA 4+ 4+ SCHWANOMA 4+ 4+

EXAMPLE XI Human Treatment Protocols

[0632] This example is concerned with human treatment protocols usingthe bispecific binding and coagulating ligands of the invention. Theseligands are contemplated for use in the clinical treatment of varioushuman cancers and even other disorders, such as benign prostatichyperplasia and rheumatoid arthritis, in which the intermediate orlonger term arrest of blood flow would be advantageous.

[0633] The bispecific ligands are considered to be particularly usefultools in anti-tumor therapy. From the data presented herein, includingthe animal studies, and the knowledge in the art regarding treatment ofLymphoma (Glennie et al., 1988), T-Cell targeting (Nolan & Kennedy,1990) and drug targeting (Paulus, 1985) appropriate doses and treatmentregimens may be straightforwardly developed.

[0634] Naturally, before wide-spread use, further animal studies andclinical trials will be conducted. The various elements of conducting aclinical trial, including patient treatment and monitoring, will beknown to those of skill in the art in light of the present disclosure.The following information is being presented as a general guideline foruse in establishing such trials.

[0635] It is contemplated that patients chosen for the study would havefailed to respond to at least one course of conventional therapy and hadto have objectively measurable disease as determined by physicalexamination, laboratory techniques, or radiographic procedures. Wheremurine monoclonal antibody portions are employed, the patients shouldhave no history of allergy to mouse immunoglobulin. Any chemotherapyshould be stopped at least 2 weeks before entry into the study.

[0636] In regard to bispecific ligand administration, it is consideredthat certain advantages will be found in the use of an indwellingcentral venous catheter with a triple lumen port. The bispecific ligandsshould be filtered, for example, using a 0.22 μm filter, and dilutedappropriately, such as with saline, to a final volume of 100 ml. Beforeuse, the test sample should also be filtered in a similar manner, andits concentration assessed before and after filtration by determiningthe A₂₈₀. The expected recovery should be within the range of 87 to 99%,and adjustments for protein loss can then be accounted for.

[0637] The bispecific ligands may be administered over a period ofapproximately 4-24 hours, with each patient receiving 2-4 number ofinfusions at 2-7 day intervals. Administration can also be performed bya steady rate of infusion over a 7 day period. The infusion given at anydose level should be dependent upon any toxicity observed. Hence, ifGrade II toxicity was reached after any single infusion, or at aparticular period of time for a steady rate infusion, further dosesshould be withheld or the steady rate infusion stopped unless toxicityimproved. Increasing doses of bispecific coagulating ligands should beadministered to groups of patients until approximately 60% of patientsshowed unacceptable Grade III or IV toxicity in any category. Doses thatare ⅔ of this value could be defined as the safe dose.

[0638] Physical examination, tumor measurements, and laboratory testsshould, of course, be performed before treatment and at intervals up to1 month later. Laboratory tests should include complete blood counts,serum creatinine, creatine kinase, electrolytes, urea, nitrogen, SGOT,bilirubin, albumin, and total serum protein. Serum samples taken up to60 days after treatment should be evaluated by radioimmunoassay for thepresence of the intact bispecific ligand or components thereof andantibodies against any or both portions of the ligand. Immunologicalanalyses of sera, using any standard assay such as, for example, anELISA or RIA, will allow the pharmacokinetics and clearance of thetherapeutic agent to be evaluated.

[0639] To evaluate the anti-tumor responses, it is contemplated that thepatients should be examined at 48 hours to 1 week and again at 30 daysafter the last infusion. When palpable disease was present, twoperpendicular diameters of all masses should be measured daily duringtreatment, within 1 week after completion of therapy, and at 30 days. Tomeasure nonpalpable disease, serial CT scans could be performed at 1-cmintervals throughout the chest, abdomen, and pelvis at 48 hours to 1week and again at 30 days. Tissue samples should also be evaluatedhistologically, and/or by flow cytometry, using biopsies from thedisease sites or even blood or fluid samples if appropriate.

[0640] Clinical responses may be defined by acceptable measure. Forexample, a complete response may be defined by the disappearance of allmeasurable tumor 1 month after treatment. Whereas a partial response maybe defined by a 50% or greater reduction of the sum of the products ofperpendicular diameters of all evaluable tumor nodules 1 month aftertreatment, with no tumor sites showing enlargement. Similarly, a mixedresponse may be defined by a reduction of the product of perpendiculardiameters of all measurable lesions by 50% or greater 1 month aftertreatment, with progression in one or more sites.

[0641] All of the compositions and methods disclosed and claimed hereincan be made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe composition, methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

References

[0642] The following references, to the extent that they provideexemplary procedural or other details supplementary to those set forthherein, are specifically incorporated herein by reference.

[0643] Abbassi et al., J Clin Invest, 92(6):2719-30, 1993.

[0644] Abraham et al., Science, 233:545-548, 1986.

[0645] Abrams & Oldham, Monoclonal antibody therapy of human cancer,Foon & Morgan (Eds.), Martinus Nijhoff Publishing, Boston, pp. 103-120,1985.

[0646] Adams et al., Cancer Res., 43:6297, 1983.

[0647] Adelman et al., DNA 2:183, 1983.

[0648] Alvarez et al., Modern Pathology, 5(3):303-307, 1992.

[0649] Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,1988.

[0650] Arklie et al., Int. J. Cancer, 28:23, 1981.

[0651] Ashall et al., Lancet, 2(8288):7-10, 1982.

[0652] Atkinson et al., Cancer Res., 62:6820, 1982.

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1 32 1 27 DNA Unknown SYNTHETIC 1 gtcatgccat ggcctcaggc actacaa 27 2 32DNA Unknown SYNTHETIC 2 tgacaagctt attctctgaa ttcccccttt ct 32 3 47 DNAUnknown SYNTHETIC 3 gtcatgccat ggccctggtg cctcgtgctt ctggcactac aaatact47 4 38 DNA Unknown SYNTHETIC 4 gtcatgccat ggcctgctca ggcactacaaatactgtg 38 5 50 DNA Unknown SYNTHETIC 5 gtcatgccat ggccctggtgcctcgtggtt cttgcggcac tacaaatact 50 6 35 DNA Unknown SYNTHETIC 6tgacaagctt agcattctct gaattccccc tttct 35 7 50 DNA Unknown SYNTHETIC 7gtcatgccat ggccctggtg cctcgtggtt gcggaggcgg tggatcaggc 50 8 36 DNAUnknown SYNTHETIC 8 agtatttgta gtgcctgagg atccgccacc tccact 36 9 45 DNAUnknown SYNTHETIC 9 ggaggcggtg gatcaggcgg tggaggtagt ggaggtggcg gatcc 4510 17 DNA Unknown SYNTHETIC 10 gtcatgccat ggccctg 17 11 32 DNA UnknownSYNTHETIC 11 tgacaagctt attctctgaa ttcccccttt ct 32 12 50 DNA UnknownSYNTHETIC 12 gtcatgccat ggccctggtg cctcgtggtt cttgcggcac tacaaatact 5013 53 DNA Unknown SYNTHETIC 13 cgcggatcca ccgccaccag atccaccgcctccttctctg aattcccctt tct 53 14 44 DNA Unknown SYNTHETIC 14 cgcggatccggcggtggagg ctcttcaggc actacaaata ctgt 44 15 31 DNA Unknown SYNTHETIC 15tgacaagctt attctctgaa ttcccctttc t 31 16 50 DNA Unknown SYNTHETIC 16gtcatgccat ggccctggtg cctcgtggtt cttgcggcac tacaaatact 50 17 31 DNAUnknown SYNTHETIC 17 tgacaagctt attctctgaa ttcccctttc t 31 18 44 DNAUnknown SYNTHETIC 18 gtcatgccat ggccctggtg cctcgtggtt gcactacaaa tact 4419 34 DNA Unknown SYNTHETIC 19 tgacaagctt agcattctct gaattcccct ttct 3420 19 DNA Unknown SYNTHETIC 20 caagttcagc caagaaaac 19 21 36 DNA UnknownSYNTHETIC 21 acactttatt atcggaaatc ttcagcttca ggaaag 36 22 657 DNA Homosapiens 22 tcaggcacta caaatactgt ggcagcatat aatttaactt ggaaatcaactaatttcaag 60 acaattttgg agtgggaacc caaacccgtc aatcaagtct acactgttcaaataagcact 120 aagtcaggag attggaaaag caaatgcttt tacacaacag acacagagtgtgacctcacc 180 gacgagattg tgaaggatgt gaagcagacg tacttggcac gggtcttctcctacccggca 240 gggaatgtgg agagcaccgg ttctgctggg gagcctctgt atgagaactccccagagttc 300 acaccttacc tggagacaaa cctcggacag ccaacaattc agagttttgaacaggtggga 360 acaaaagtga atgtgaccgt agaagatgaa cggactttag tcagaaggaacaacactttc 420 ctaagcctcc gggatgtttt tggcaaggac ttaatttata cactttattattggaaatct 480 tcaagttcag gaaagaaaac agccaaaaca aacactaatg agtttttgattgatgtggat 540 aaaggagaaa actactgttt cagtgttcaa gcagtgattc cctcccgaacagttaaccgg 600 aagagtacag acagcccggt agagtgtatg ggccaggaga aaggggaattcagagaa 657 23 219 PRT Homo sapiens 23 Ser Gly Thr Thr Asn Thr Val AlaAla Tyr Asn Leu Thr Trp Lys Ser 1 5 10 15 Thr Asn Phe Lys Thr Ile LeuGlu Trp Glu Pro Lys Pro Val Asn Gln 20 25 30 Val Tyr Thr Val Gln Ile SerThr Lys Ser Gly Asp Trp Lys Ser Lys 35 40 45 Cys Phe Tyr Thr Thr Asp ThrGlu Cys Asp Leu Thr Asp Glu Ile Val 50 55 60 Lys Asp Val Lys Gln Thr TyrLeu Ala Arg Val Phe Ser Tyr Pro Ala 65 70 75 80 Gly Asn Val Glu Ser ThrGly Ser Ala Gly Glu Pro Leu Tyr Glu Asn 85 90 95 Ser Pro Glu Phe Thr ProTyr Leu Glu Thr Asn Leu Gly Gln Pro Thr 100 105 110 Ile Gln Ser Phe GluGln Val Gly Thr Lys Val Asn Val Thr Val Glu 115 120 125 Asp Glu Arg ThrLeu Val Arg Arg Asn Asn Thr Phe Leu Ser Leu Arg 130 135 140 Asp Val PheGly Lys Asp Leu Ile Tyr Thr Leu Tyr Tyr Trp Lys Ser 145 150 155 160 SerSer Ser Gly Lys Lys Thr Ala Lys Thr Asn Thr Asn Glu Phe Leu 165 170 175Ile Asp Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser Val Gln Ala Val 180 185190 Ile Pro Ser Arg Thr Val Asn Arg Lys Ser Thr Asp Ser Pro Val Glu 195200 205 Cys Met Gly Gln Glu Lys Gly Glu Phe Arg Glu 210 215 24 1947 DNAHomo sapiens 24 tgcagctgcc tggctgcctg gccctggctg ccctgtgtag ccttgtgcacagccagcatg 60 tgttcctggc tcctcagcaa gcacggtcgc tgctccagcg ggtccggcgagccaacacct 120 tcttggagga ggtgcgcaag ggcaacctag agcgagagtg cgtggaggagacgtgcagct 180 acgaggaggc cttcgaggct ctggagtcct ccacggctac ggatgtgttctgggccaagt 240 acacagcttg tgagacagcg aggacgcctc gagataagct tgctgcatgtctggaaggta 300 actgtgctga gggtctgggt acgaactacc gagggcatgt gaacatcacccggtcaggca 360 ttgagtgcca gctatggagg agtcgctacc cacataagcc tgaaatcaactccactaccc 420 atcctggggc cgacctacag gagaatttct gccgcaaccc cgacagcagcaacacgggac 480 cctggtgcta cactacagac cccaccgtga ggaggcagga atgcagcatccctgtctgtg 540 gccaggatca agtcactgta gcgatgactc cacgctccga aggctccagtgtgaatctgt 600 cacctccatt ggagcagtgt gtccctgatc gggggcagca gtaccaggggcgcctggcgg 660 tgaccacaca tgggctcccc tgcctggcct gggccagcgc acaggccaaggccctgagca 720 agcaccagga cttcaactca gctgtgcagc tggtggagaa cttctgccgcaacccagacg 780 gggatgagga gggcgtgtgg tgctatgtgg ccgggaagcc tggcgactttgggtactgcg 840 acctcaacta ttgtgaggag gccgtggagg aggagacagg agatgggctggatgaggact 900 cagacagggc catcgaaggg cgtaccgcca caagtgagta ccagactttcttcaatccga 960 ggacctttgg ctcgggagag gcagactgtg ggctgcgacc tctgttcgagaagaagtcgc 1020 tggaggacaa aaccgaaaga gagctcctgg aatcctacat cgacgggcgcattgtggagg 1080 gctcggatgc agagatcggc atgtcacctt ggcaggtgat gcttttccggaagagtcccc 1140 aggagctgct gtgtggggcc agcctcatca gtgaccgctg ggtcctcaccgccgcccact 1200 gcctcctgta cccgccctgg gacaagaact tcaccgagaa tgaccttctggtgcgcattg 1260 gcaagcactc ccgcaccagg tacgagcgaa acattgaaaa gatatccatgttggaaaaga 1320 tctacatcca ccccaggtac aactggcggg agaacctgga ccgggacattgccctgatga 1380 agctgaagaa gcctgttgcc ttcagtgact acattcaccc tgtgtgtctgcccgacaggg 1440 agacggcagc cagcttgctc caggctggat acaaggggcg ggtgacaggctggggcaacc 1500 tgaaggagac gtggacagcc aacgttggta aggggcagcc cagtgtcctgcaggtggtga 1560 acctgcccat tgtggagcgg ccggtctgca aggactccac ccggatccgcatcactgaca 1620 acatgttctg tgctggttac aagcctgatg aagggaaacg aggggatgcctgtgaaggtg 1680 acagtggggg accctttgtc atgaagagcc cctttaacaa ccgctggtatcaaatgggca 1740 tcgtctcatg gggtgaaggc tgtgaccggg atgggaaata tggcttctacacacatgtgt 1800 tccgcctgaa gaagtggata cagaaggtca ttgatcagtt tggagagtagggggccactc 1860 atattctggg ctcctggaac caatcccgtg aaagaattat ttttgtgtttctaaaactat 1920 ggttcccaat aaaagtgact ctcagcg 1947 25 2462 DNA Homosapiens 25 tcaacaggca ggggcagcac tgcagagatt tcatcatggt ctcccaggccctcaggctcc 60 tctgccttct gcttgggctt cagggctgcc tggctgcagg cggggtcgctaaggcctcag 120 gaggagaaac acgggacatg ccgtggaagc cggggcctca cagagtcttcgtaacccagg 180 aggaagccca cggcgtcctg caccggcgcc ggcgcgccaa cgcgttcctggaggagctgc 240 ggccgggctc cctggagagg gagtgcaagg aggagcagtg ctccttcgaggaggcccggg 300 agatcttcaa ggacgcggag aggacgaagc tgttctggat ttcttacagtgatggggacc 360 agtgtgcctc aagtccatgc cagaatgggg gctcctgcaa ggaccagctccagtcctata 420 tctgcttctg cctccctgcc ttcgagggcc ggaactgtga gacgcacaaggatgaccagc 480 tgatctgtgt gaacgagaac ggcggctgtg agcagtactg cagtgaccacacgggcacca 540 agcgctcctg tcggtgccac gaggggtact ctctgctggc agacggggtgtcctgcacac 600 ccacagttga atatccatgt ggaaaaatac ctattctaga aaaaagaaatgccagcaaac 660 cccaaggccg aattgtgggg ggcaaggtgt gccccaaagg ggagtgtccatggcaggtcc 720 tgttgttggt gaatggagct cagttgtgtg gggggaccct gatcaacaccatctgggtgg 780 tctccgcggc ccactgtttc gacaaaatca agaactggag gaacctgatcgcggtgctgg 840 gcgagcacga cctcagcgag cacgacgggg atgagcagag ccggcgggtggcgcaggtca 900 tcatccccag cacgtacgtc ccgggcacca ccaaccacga catcgcgctgctccgcctgc 960 accagcccgt ggtcctcact gaccatgtgg tgcccctctg cctgcccgaacggacgttct 1020 ctgagaggac gctggccttc gtgcgcttct cattggtcag cggctggggccagctgctgg 1080 accgtggcgc cacggccctg gagctcatgg tgctcaacgt gccccggctgatgacccagg 1140 actgcctgca gcagtcacgg aaggtgggag actccccaaa tatcacggagtacatgttct 1200 gtgccggcta ctcggatggc agcaaggact cctgcaaggg ggacagtggaggcccacatg 1260 ccacccacta ccggggcacg tggtacctga cgggcatcgt cagctggggccagggctgcg 1320 caaccgtggg ccactttggg gtgtacacca gggtctccca gtacatcgagtggctgcaaa 1380 agctcatgcg ctcagagcca cgcccaggag tcctcctgcg agccccatttccctagccca 1440 gcagccctgg cctgtggaga gaaagccaag gctgcgtcga actgtcctggcaccaaatcc 1500 catatattct tctgcagtta atggggtaga ggagggcatg ggagggagggagaggtgggg 1560 agggagacag agacagaaac agagagagac agagacagag agagactgagggagagactc 1620 tgaggacatg gagagagact caaagagact ccaagattca aagagactaatagagacaca 1680 gagatggaat agaaaagatg agaggcagag gcagacaggc gctggacagaggggcagggg 1740 agtgccaagg ttgtcctgga ggcagacagc ccagctgagc ctccttacctcccttcagcc 1800 aagccccacc tgcacgtgat ctgctggccc tcaggctgct gctctgccttcattgctgga 1860 gacagtagag gcatgaacac acatggatgc acacacacac acgccaatgcacacacacag 1920 agatatgcac acacacggat gcacacacag atggtcacac agagatacgcaaacacaccg 1980 atgcacacgc acatagagat atgcacacac agatgcacac acagatatacacatggatgc 2040 acgcacatgc caatgcacgc acacatcagt gcacacggat gcacagagatatgcacacac 2100 cgatgtgcgc acacacagat atgcacacac atggatgagc acacacacaccaagtgcgca 2160 cacacaccga tgtacacaca cagatgcaca cacagatgca cacacaccgatgctgactcc 2220 atgtgtgctg tcctctgaag gcggttgttt agctctcact tttctggttcttatccatta 2280 tcatcttcac ttcagacaat tcagaagcat caccatgcat ggtggcgaatgcccccaaac 2340 tctcccccaa atgtatttct cccttcgctg ggtgccgggc tgcacagactattccccacc 2400 tgcttcccag cttcacaata aacggctgcg tctcctccgc acacctgtggtgcctgccac 2460 cc 2462 26 1437 DNA Homo sapiens 26 atgcagcgcgtgaacatgat catggcagaa tcaccaagcc tcatcaccat ctgcctttta 60 ggatatctactcagtgctga atgtacagtt tttcttgatc atgaaaacgc caacaaaatt 120 ctgaatcggccaaagaggta taattcaggt aaattggaag agtttgttca agggaacctt 180 gagagagaatgtatggaaga aaagtgtagt tttgaagaac cacgagaagt ttttgaaaac 240 actgaaaagacaactgaatt ttggaagcag tatgttgatg gagatcagtg tgagtccaat 300 ccatgtttaaatggcggcag ttgcaaggat gacattaatt cctatgaatg ttggtgtccc 360 tttggatttgaaggaaagaa ctgtgaatta gatgtaacat gtaacattaa gaatggcaga 420 tgcgagcagttttgtaaaaa tagtgctgat aacaaggtgg tttgctcctg tactgaggga 480 tatcgacttgcagaaaacca gaagtcctgt gaaccagcag tgccatttcc atgtggaaga 540 gtttctgtttcacaaacttc taagctcacc cgtgctgagg ctgtttttcc tgatgtggac 600 tatgtaaatcctactgaagc tgaaaccatt ttggataaca tcactcaagg cacccaatca 660 tttaatgacttcactcgggt tgttggtgga gaagatgcca aaccaggtca attcccttgg 720 caggttgttttgaatggtaa agttgatgca ttctgtggag gctctatcgt taatgaaaaa 780 tggattgtaactgctgccca ctgtgttgaa actggtgtta aaattacagt tgtcgcaggt 840 gaacataatattgaggagac agaacataca gagcaaaagc gaaatgtgat tcgagcaatt 900 attcctcaccacaactacaa tgcagctatt aataagtaca accatgacat tgcccttctg 960 gaactggacgaacccttagt gctaaacagc tacgttacac ctatttgcat tgctgacaag 1020 gaatacacgaacatcttcct caaatttgga tctggctatg taagtggctg ggcaagagtc 1080 ttccacaaagggagatcagc tttagttctt cagtacctta gagttccact tgttgaccga 1140 gccacatgtcttcgatctac aaagttcacc atctataaca acatgttctg tgctggcttc 1200 catgaaggaggtagagattc atgtcaagga gatagtgggg gaccccatgt tactgaagtg 1260 gaagggaccagtttcttaac tggaattatt agctggggtg aagagtgtgc aatgaaaggc 1320 aaatatggaatatataccaa ggtatcccgg tatgtcaact ggattaagga aaaaacaaag 1380 ctcacttaatgaaagatgga tttccaaggt taattcattg gaattgaaaa ttaacag 1437 27 1126 DNAHomo sapiens 27 ggattcgaag gcaaaaactg tgaattattc acacggaagc tctgcagcctggacaacggg 60 gactgtgacc agttctgcca cgaggaacag aactctgtgg tgtgctcctgcgcccgcggg 120 tacaccctgg ctgacaacgg caaggcctgc attcccacag ggccctacccctgtgggaaa 180 cagaccctgg aacgcaggaa gaggtcagtg gcccaggcca ccagcagcagcggggaggcc 240 cctgacagca tcacatggaa gccatatgat gcagccgacc tggaccccaccgagaacccc 300 ttcgacctgc ttgacttcaa ccagacgcag cctgagaggg gcgacaacaacctcaccagg 360 atcgtgggag gccaggaatg caaggacggg gagtgtccct ggcaggccctgctcatcaat 420 gaggaaaacg agggtttctg tggtggaacc attctgagcg agttctacatcctaacggca 480 gcccactgtc tctaccaagc caagagattc gaaggggacc ggaacacggagcaggaggag 540 ggcggtgagg cggtgcacga ggtggaggtg gtcatcaagc acaaccggttcacaaaggag 600 acctatgact tcgacatcgc cgtgctccgg ctcaagaccc ccatcaccttccgcatgaac 660 gtggcgcctg cctgcctccc cgagcgtgac tgggccgagt ccacgctgatgacgcagaag 720 acggggattg tgagcggctt cgggcgcacc cacgagaagg gccggcagtccaccaggctc 780 aagatgctgg aggtgcccta cgtggaccgc aacagctgca agctgtccagcagcttcatc 840 atcacccaga acatgttctg tgccggctac gacaccaagc aggaggatgcctgccagggg 900 gacagcgggg gcccgcacgt cacccgcttc aaggacacct acttcgtgacaggcatcgtc 960 agctggggag agggctgtgc ccgtaagggg aagtacggga tctacaccaaggtcaccgcc 1020 ttcctcaagt ggatcgacag gtccatgaaa accaggggct tgcccaaggccaagagccat 1080 gccccggagg tcataacgtc ctctccatta aagtgagatc ccactc 112628 45 DNA Unknown SYNTHETIC 28 gaagaaggga tcctggtgcc tcgtggttctggcactacaa atact 45 29 30 DNA Unknown SYNTHETIC 29 ctggcctcaa gcttaacggaattcaccttt 30 30 25 PRT Cavia porcellus 30 Ala Pro Met Ala Glu Gly GluGln Lys Pro Arg Glu Val Val Lys Phe 1 5 10 15 Met Asp Val Tyr Lys ArgSer Tyr Cys 20 25 31 26 PRT Homo sapiens 31 Ala Pro Met Ala Glu Gly GlyGly Gln Asn His His Glu Val Val Lys 1 5 10 15 Phe Met Asp Val Tyr GlnArg Ser Tyr Cys 20 25 32 25 PRT Cavia porcellus 32 Met Ala Ala Gly SerIle Thr Thr Leu Pro Ala Leu Pro Glu Gly Gly 1 5 10 15 Asp Gly Gly AlaPhe Ala Pro Gly Cys 20 25

What is claimed is:
 1. A binding ligand comprising: (a) a first bindingregion that binds to a diseased cell, a component of disease-associatedvasculature or a component of disease-associated stroma; the firstbinding region operatively linked to (b) a coagulation factor or asecond binding region that binds to a coagulation factor.
 2. The bindingligand of claim 1, wherein said first binding region comprises an IgGantibody, an IgM antibody or an antigen binding region of an antibody.3. The binding ligand of claim 2, wherein said first binding regioncomprises an scFv, Fv, Fab′, Fab or F(ab′)₂ fragment of an antibody. 4.The binding ligand of claim 2, wherein said first binding regioncomprises an antigen binding region of an antibody that binds to a tumorcell, a component of tumor vasculature or a component of tumor stroma.5. The binding ligand of claim 4, wherein said first binding regioncomprises an antigen binding region of an antibody that binds to a cellsurface antigen of a tumor cell.
 6. The binding ligand of claim 5,wherein said first binding region comprises an antigen binding region ofan antibody that binds to the cell surface tumor antigen p185^(HER2),milk mucin core protein, TAG-72, Lewis a, carcinoembryonic antigen (CEA)or a tumor-associated antigen that binds to an antibody selected fromthe group consisting of 9.2.27, OV-TL3, MOv18, B3, KS1/4, 260F9 andD612.
 7. The binding ligand of claim 4, wherein said first bindingregion comprises an antigen binding region of an antibody that binds toa component of tumor vasculature.
 8. The binding ligand of claim 7,wherein said first binding region comprises an antigen binding region ofan antibody that binds to a tumor vasculature cell surface receptor. 9.The binding ligand of claim 8, wherein said first binding regioncomprises an antigen binding region of an antibody that binds to an MHCClass II protein, a VEGF/VPF receptor, an FGF receptor, a TGFβ receptor,a TIE, VCAM-1, P-selectin, E-selectin, α_(v)β₃ integrin, pleiotropin,endosialin or endoglin.
 10. The binding ligand of claim 9, wherein saidfirst binding region comprises an antigen binding region of an antibodythat binds to endoglin.
 11. The binding ligand of claim 10, wherein saidfirst binding region comprises an antigen binding region of an antibodythat binds to the same epitope as the monoclonal antibody TEC-4 or themonoclonal antibody TEC-11.
 12. The binding ligand of claim 9, whereinsaid first binding region comprises an antigen binding region of anantibody that binds to a VEGF receptor.
 13. The binding ligand of claim12, wherein said first binding region comprises an antigen bindingregion of an antibody that binds to the same epitope as the monoclonalantibody 3E11, 3E7, 5G6, 4D8 or 10B10.
 14. The binding ligand of claim12, wherein said first binding region comprises an antigen bindingregion of an antibody that binds to the same epitope as the monoclonalantibody TEC-110.
 15. The binding ligand of claim 7, wherein said firstbinding region comprises an antigen binding region of an antibody thatbinds to a ligand that binds to a tumor vasculature cell surfacereceptor.
 16. The binding ligand of claim 15, wherein said first bindingregion comprises an antigen binding region of an antibody that binds toVEGF/VPF, FGF, TGFβ, a ligand that binds to a TIE, a tumor-associatedfibronectin isoform, scatter factor, hepatocyte growth factor (HGF),platelet factor 4 (PF4), PDGF or TIMP.
 17. The binding ligand of claim16, wherein said first binding region comprises an antigen bindingregion of an antibody that binds to VEGF/VPF, FGF, TGFβ, a ligand thatbinds to a TIE or a tumor-associated fibronectin isoform.
 18. Thebinding ligand of claim 7, wherein said first binding region comprisesan antigen binding region of an antibody that binds to an inducibletumor vasculature component.
 19. The binding ligand of claim 18, whereinsaid first binding region comprises an antigen binding region of anantibody that binds to a tumor vasculature component inducible by acoagulant.
 20. The binding ligand of claim 19, wherein said firstbinding region comprises an antigen binding region of an antibody thatbinds to a tumor vasculature component inducible by thrombin.
 21. Thebinding ligand of claim 20, wherein said first binding region comprisesan antigen binding region of an antibody that binds to P-selectin,E-selectin, PDGF or ICAM-1.
 22. The binding ligand of claim 18, whereinsaid first binding region comprises an antigen binding region of anantibody that binds to a tumor vasculature component inducible by acytokine.
 23. The binding ligand of claim 22, wherein said first bindingregion comprises an antigen binding region of an antibody that binds toa tumor vasculature component inducible by a cytokine released bymonocytes, macrophages, mast cells, helper T cells, CD8-positive T-cellsor NK cells.
 24. The binding ligand of claim 22, wherein said firstbinding region comprises an antigen binding region of an antibody thatbinds to a tumor vasculature component inducible by the cytokine IL-1,IL-4, TNF-α, TNF-β or IFN-γ.
 25. The binding ligand of claim 22, whereinsaid first binding region comprises an antigen binding region of anantibody that binds to E-selectin, VCAM-1, ICAM-1, endoglin or an MHCClass II antigen.
 26. The binding ligand of claim 25, wherein said firstbinding region comprises an antigen binding region of an antibody thatbinds to E-selectin.
 27. The binding ligand of claim 25, wherein saidfirst binding region comprises an antigen binding region of an antibodythat binds to an MHC Class II antigen.
 28. The binding ligand of claim7, wherein said first binding region comprises an antigen binding regionof an antibody that binds to a ligand:receptor complex but does not bindto the ligand or the factor receptor when the ligand or receptor is notin the ligand:receptor complex.
 29. The binding ligand of claim 28,wherein said first binding region comprises an antigen binding region ofan antibody that binds to the same epitope as the monoclonal antibody2E5, 3E5 or 4E5.
 30. The binding ligand of claim 4, wherein said firstbinding region comprises an antigen binding region of an antibody thatbinds to a component of tumor stroma.
 31. The binding ligand of claim30, wherein said first binding region comprises an antigen bindingregion of an antibody that binds to tenascin.
 32. The binding ligand ofclaim 30, wherein said first binding region comprises an antigen bindingregion of an antibody that binds to a basement membrane component. 33.The binding ligand of claim 30, wherein said first binding regioncomprises an antigen binding region of an antibody that binds to anactivated platelet.
 34. The binding ligand of claim 30, wherein saidfirst binding region comprises an antigen binding region of an antibodythat binds to an inducible tumor stroma component.
 35. The bindingligand of claim 34, wherein said first binding region comprises anantigen binding region of an antibody that binds to a tumor stromacomponent inducible by a coagulant.
 36. The binding ligand of claim 35,wherein said first binding region comprises an antigen binding region ofan antibody that binds to a tumor stroma component inducible bythrombin.
 37. The binding ligand of claim 36, wherein said first bindingregion comprises an antigen binding region of an antibody that binds toRIBS.
 38. The binding ligand of claim 1, wherein said first bindingregion comprises a ligand or receptor that binds to a diseased cell orto a component of disease-associated vasculature.
 39. The binding ligandof claim 38, wherein said first binding region comprises a ligand thatbinds to a tumor cell surface receptor or a soluble binding domain of areceptor that binds to a ligand that binds to a tumor cell surfacemolecule.
 40. The binding ligand of claim 38, wherein said first bindingregion comprises a ligand or receptor that binds to a component of tumorvasculature.
 41. The binding ligand of claim 40, wherein said firstbinding region comprises a ligand that binds to a tumor vasculatureendothelial cell surface receptor.
 42. The binding ligand of claim 41,wherein said first binding region comprises VEGF/VPF, FGF, TGFβ, aligand that binds to a TIE, a tumor-associated fibronectin isoform,scatter factor, hepatocyte growth factor (HGF), platelet factor 4 (PF4),PDGF or TIMP.
 43. The binding ligand of claim 42, wherein said firstbinding region comprises VEGF/VPF.
 44. The binding ligand of claim 42,wherein said first binding region comprises FGF.
 45. The binding ligandof claim 40, wherein said first binding region comprises a solublebinding domain of a receptor that binds to a ligand that binds to atumor vasculature endothelial cell surface receptor.
 46. The bindingligand of claim 45, wherein said first binding region comprises asoluble binding domain of a VEGF/VPF receptor.
 47. The binding ligand ofclaim 1, wherein said first binding region is operatively linked to acoagulation factor.
 48. The binding ligand of claim 47, wherein saidcoagulation factor comprises the vitamin K-dependent coagulant FactorII/IIa, Factor VII/VIIa, Factor IX/IXa or Factor X/Xa.
 49. The bindingligand of claim 48, wherein said coagulation factor comprises a vitaminK-dependent coagulation factor lacking the Gla modification.
 50. Thebinding ligand of claim 49, wherein said coagulation factor is preparedby expressing a vitamin K-dependent coagulation factor-encoding gene ina procaryotic host cell.
 51. The binding ligand of claim 49, whereinsaid coagulation factor is prepared by treating the vitamin K-dependentcoagulation factor protein to remove or alter the corresponding Glutamicacid residues.
 52. The binding ligand of claim 49, wherein saidcoagulation factor is prepared by preparing an engineered coagulationfactor gene that encodes a vitamin K-dependent coagulation factorlacking the corresponding Glutamic acid residues and expressing saidengineered gene in a recombinant host cell.
 53. The binding ligand ofclaim 47, wherein said coagulation factor comprises Tissue Factor or aTissue Factor derivative.
 54. The binding ligand of claim 53, whereinsaid coagulation factor comprises a mutant Tissue Factor deficient inthe ability to activate Factor VII.
 55. The binding ligand of claim 54,wherein said coagulation factor comprises a Tissue Factor that includesa mutation in the amino acid region between about position 157 and aboutposition
 167. 56. The binding ligand of claim 55, wherein saidcoagulation factor comprises a mutant Tissue Factor wherein Trp atposition 158 is changed to Arg; wherein Ser at position 162 is changedto Ala; wherein Gly at position 164 is changed to Ala; or wherein Trp atposition 158 is changed to Arg and Ser at position 162 is changed toAla.
 57. The binding ligand of claim 53, wherein said coagulation factorcomprises truncated Tissue Factor.
 58. The binding ligand of claim 57,wherein said coagulation factor comprises dimeric truncated TissueFactor.
 59. The binding ligand of claim 47, wherein said coagulationfactor comprises Russell's viper venom Factor X activator.
 60. Thebinding ligand of claim 47, wherein said coagulation factor comprises aplatelet-activating compound.
 61. The binding ligand of claim 60,wherein said coagulation factor comprises thromboxane A₂ or thromboxaneA₂ synthase.
 62. The binding ligand of claim 47, wherein saidcoagulation factor comprises an inhibitor of fibrinolysis.
 63. Thebinding ligand of claim 62, wherein said coagulation factor comprisesα2-antiplasmin.
 64. The binding ligand of claim 1, wherein said firstbinding region is operatively linked to a second binding region thatbinds to a coagulation factor.
 65. The binding ligand of claim 64,further comprising a coagulation factor bound to said second bindingregion.
 66. The binding ligand of claim 64, wherein said second bindingregion comprises an antigen binding region of an antibody that binds toa coagulation factor.
 67. The binding ligand of claim 66, wherein saidsecond binding region comprises an IgG antibody, an IgM antibody, or ascFv, Fv, Fab′, Fab or F(ab′)₂ fragment of an antibody.
 68. The bindingligand of claim 66, wherein said second binding region comprises anantigen binding region of an antibody that binds to the vitaminK-dependent coagulant Factor II/IIa, Factor VII/VIIa, Factor IX/IXa orFactor X/Xa.
 69. The binding ligand of claim 68, wherein said secondbinding region comprises an antigen binding region of an antibody thatbinds to a vitamin K-dependent coagulation factor that lacks the Glamodification.
 70. The binding ligand of claim 66, wherein said secondbinding region comprises an antigen binding region of an antibody thatbinds to Tissue Factor.
 71. The binding ligand of claim 70, wherein saidsecond binding region comprises an antigen binding region of an antibodythat binds to a mutant Tissue Factor.
 72. The binding ligand of claim70, wherein said second binding region comprises an antigen bindingregion of an antibody that binds to truncated Tissue Factor.
 73. Thebinding ligand of claim 72, wherein said second binding region comprisesan antigen binding region of an antibody that binds to dimeric truncatedTissue Factor.
 74. The binding ligand of claim 66, wherein said secondbinding region comprises an antigen binding region of an antibody thatbinds to Russell's viper venom Factor X activator, thromboxane A₂ orα2-antiplasmin.
 75. The binding ligand of claim 1, wherein said firstbinding region is operatively linked to said coagulation factor or saidsecond binding region via a covalent bond.
 76. The binding ligand ofclaim 75, wherein said first binding region is operatively linked tosaid coagulation factor or said second binding region via a chemicalcross-linker.
 77. The binding ligand of claim 75, wherein said bindingligand is a fusion protein prepared by expressing a recombinant vectorin a host cell, wherein the vector comprises, in the same reading frame,a DNA segment encoding said first binding region operatively linked to aDNA segment encoding said coagulation factor or second binding region.78. The binding ligand of claim 1, wherein said first binding region isoperatively linked to said coagulation factor or said second bindingregion using an avidin:biotin combination.
 79. The binding ligand ofclaim 1, further defined as a bispecific antibody comprising a firstantigen binding region that binds to a tumor cell, a component oftumor-associated vasculature or a component of tumor-associated stroma,the first antigen binding region operatively linked to a second antigenbinding region that binds to a coagulation factor.
 80. The bindingligand of claim 79, further defined as an IgG antibody, an IgM antibodyor an scFv, Fv, Fab′, Fab or F(ab′)₂ fragment of a bispecific antibody.81. The binding ligand of claim 79, further defined as a bispecificantibody comprising a first antigen binding region that binds to an MHCClass II protein operatively linked to a second antigen binding regionthat binds to truncated Tissue Factor.
 82. A binding ligand comprising:(a) a first binding region that binds to a tumor cell, a component oftumor-associated vasculature or a component of tumor-associated stroma;the first binding region operatively linked to (b) an engineeredcoagulation factor or a second binding region that binds to anengineered coagulation factor.
 83. The binding ligand of claim 82,wherein said engineered coagulation factor is a vitamin K-dependentcoagulant that lacks the Gla modification.
 84. The binding ligand ofclaim 82, wherein said engineered coagulation factor is a Tissue Factorconstruct comprising a first Tissue Factor or derivative operativelylinked to a second Tissue Factor or derivative.
 85. A Tissue Factorconstruct comprising a first Tissue Factor or derivative operativelylinked to a second Tissue Factor or derivative.
 86. The Tissue Factorconstruct of claim 85, comprising an operatively linked series of unitsin the sequence: a Cysteine residue, a selectively cleavable peptidelinker, a stretch of hydrophobic amino acids, a first Tissue Factor orderivative and a second Tissue Factor or derivative.
 87. Apharmaceutical composition comprising, in a pharmacologically acceptableform, a binding ligand that comprises: (a) a first binding region thatbinds to a diseased cell, a component of disease-associated vasculatureor a component of disease-associated stroma; the first binding regionoperatively linked to (b) a coagulation factor or a second bindingregion that binds to a coagulation factor.
 88. A kit comprising, insuitable container means: (a) a first pharmaceutical compositioncomprising a biological agent capable of inducing the expression of atarget antigen in disease-associated vasculature or disease-associatedstroma; and (b) a second pharmaceutical composition comprising a bindingligand that comprises (i) a first binding region that binds to aninducible target antigen of disease-associated vasculature ordisease-associated stroma, the first binding region operatively linkedto (ii) a coagulation factor or a second binding region that binds to acoagulation factor.
 89. A method for delivering a coagulant todisease-associated vasculature, comprising administering to an animalwith a disease that has a vascular component, a pharmaceuticalcomposition comprising an effective amount of a binding ligand thatcomprises: (a) a first binding region that binds to a diseased cell, acomponent of disease-associated vasculature or a component ofdisease-associated stroma, the first binding region operatively linkedto (b) coagulation factor or a second binding region that binds to acoagulation factor.
 90. A method for targeting a coagulant todisease-associated endothelial vasculature in an animal with a diseasethat has a vascular component, the method comprising the steps of: (a)inducing the expression of E-selectin, an MHC Class II molecule orP-selectin in disease-associated vascular endothelial cells; (b)preparing an antibody that binds to E-selectin, an MHC Class IIdeterminant or P-selectin; (c) linking a coagulation factor or a secondbinding region that binds to a coagulation factor to said antibody; and(d) introducing the antibody-linked coagulant into the bloodstream ofthe animal.
 91. A method for treating cancer, comprising the steps of:(a) preparing a pharmaceutical composition comprising a coagulativebinding ligand that comprises (i) a first binding region that binds to atumor cell, a component of tumor-associated vasculature or a componentof tumor-associated stroma, the first binding region operatively linkedto (ii) a coagulation factor or a second binding region that binds to acoagulation factor; and (b) administering said pharmaceuticalcomposition to an animal with a vascularized tumor in an amounteffective to promote blood coagulation in the vasculature of said tumor.